Improved microbial growth medium

Abstract

The compositions, devices and methods described herein relate to the discovery that the detection time of yeast and microbial contaminants in a microbial growth medium can be improved by controlling the concentration of iron and copper.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 482,760, filed Feb. 1, 2023, which is hereby incorporated by reference in its entirety.

[0003] Field

[0004] The embodiments described herein generally relate to enhanced microbial growth media that provide increased growth rates and / or shortened detection times for yeast and / or other microorganisms.

[0005] Background

[0006] Existing microbial growth media contain a yeast nitrogen base that does not have amino acids, and the microbial media include unspecified amounts of iron, copper, and other nutrients. At the volumes required for full - scale production, the presence of the yeast nitrogen base causes precipitation of iron in the media. The uncertain concentrations of iron and copper in existing microbial growth media delay the detection times of certain microorganisms. Accordingly, there is a need in the art for improved microbial growth media.

[0007] Summary

[0008] The compositions, devices, and methods described herein relate to the discovery that by controlling the concentrations of iron and optionally copper, the growth rates and / or detection times of yeast and / or other microorganisms in microbial growth media can be improved.

[0009] The embodiments provided herein include the following numbered embodiments:

[0010] 1. A microbial growth medium that provides an increased growth rate of Candida yeast, the microbial growth medium comprising:

[0011] iron at a concentration of from about 1 μM to about 400 μM; and

[0012] optionally copper at a concentration not exceeding about 700 μM;

[0013] wherein the growth rate of Candida yeast cultured in the microbial growth medium is increased compared to the same microbial growth medium without the iron and copper.

[0014] 2. The microbial growth medium according to embodiment 1, wherein the time to detection (TTD) of Candida yeast cultured in the microbial growth medium is shortened compared to the same microbial growth medium without the iron and copper.

[0015] 3. The microbial growth medium according to any one of the foregoing embodiments, wherein the shortened TTD of Candida cultured in the microbial growth medium is determined by:

[0016] Add 0.5 mL, 3 mL, and 10 mL of blood to culture vessels containing the microbial growth medium with and without the iron and copper;

[0017] Inoculate the culture vessels with 0.1 mL of Candida culture to provide 10 - 100 CFU of Candida per vessel;

[0018] Cultivate the Candida at about 30°C to 37°C for up to 120 hours;

[0019] Monitor the signal indicating the presence of Candida in the culture vessels; and

[0020] Determine the TTD of the Candida in the microbial growth medium with and without the iron and copper.

[0021] 4. The microbial growth medium according to any one of the foregoing embodiments, wherein the Candida is Candida albicans ( Candida albicans ) or Candida glabrata ( Candida glabrata ).

[0022] 5. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium contains or contains about 0 g / L, 0.01 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1.0 g / L of yeast nitrogen base, or a yeast nitrogen base within a range defined by any two of the foregoing values.

[0023] 6. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium contains less than 0.01% w / v of yeast nitrogen base.

[0024] 7. The microbial growth medium according to any one of the foregoing embodiments, wherein the yeast nitrogen base does not contain histidine, methionine, and tryptophan.

[0025] 8. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium comprises or consists essentially of about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 28 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 170 μM, 175 μM, 200 μM, 225 μM, 250 μM, 275 μM, 300 μM, 325 μM, 350 μM or 400 μM of iron, or a range defined by any two of the foregoing values, optionally 1 μM to 400 μM, 1 μM to 350 μM, 1 μM to 200 μM, 1 μM to 100 μM, 1 μM to 50 μM, 2 μM to 400 μM, 2 μM to 350 μM, 25 μM to 400 μM, 25 μM to 325 μM, 25 μM to 200 μM, 25 μM to 100 μM or 25 μM to 75 μM of iron.

[0026] 9. The microbial growth medium according to any one of the foregoing embodiments, wherein the concentration of iron is about 28 μM.

[0027] 10. The microbial growth medium according to any one of the foregoing embodiments, wherein the concentration of iron is about 100 μM.

[0028] 11. The microbial growth medium according to any one of the foregoing embodiments, wherein the concentration of iron is about 170 μM.

[0029] 12. The microbial growth medium according to any one of the foregoing embodiments, wherein the iron is ferric iron.

[0030] 13. The microbial growth medium according to any one of the foregoing embodiments, wherein the iron is provided as ammonium ferric citrate, ferric chloride, ferric sulfate, ferric nitrate.

[0031] 14. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium comprises or consists essentially of about 0 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 650 μM or 700 μM of copper, or a range defined by any two of the foregoing values, optionally 0 μM to 700 μM, 0 μM to 650 μM, 0 μM to 500 μM, 0 μM to 300 μM, 0 μM to 100 μM, 0 μM to 50 μM, 0 μM to 25 μM, 0.1 μM to 100 μM, 0.1 μM to 50 μM, 0.1 μM to 25 μM, 0.5 μM to 650 μM, 0.5 μM to 300 μM, 0.5 μM to 100 μM, 0.5 μM to 50 μM or 0.5 μM to 25 μM of copper.

[0032] 15. The microbial growth medium according to any one of the foregoing embodiments, wherein the concentration of copper is about 3 μM.

[0033] 16. The microbial growth medium according to any one of the foregoing embodiments, wherein the copper is divalent copper.

[0034] 17. The microbial growth medium according to any one of the foregoing embodiments, wherein the copper is provided as copper sulfate, copper chloride, copper nitrate, copper bromide, copper chlorate and / or copper(II) gluconate.

[0035] 18. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium is an aqueous liquid growth medium, and the aqueous liquid growth medium further comprises sucrose, dextrose, D-trehalose, yeast extract, L-glutamic acid, tryptic soy broth (TSB), sodium polyanetholesulfonate (SPS), menaquinone, pyridoxal hydrochloride, ferulic acid, sodium hydroxide, ascorbic acid, L-cysteine, hemin.

[0036] 19. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium comprises, in water or in an aqueous solution, about 1.0 g / L to 10.0 g / L yeast extract, 0.15 g / L to 1.5 g / L sucrose, 1.0 g / L to 10.0 g / L dextrose, 1.0 g / L to 10.0 g / L D-trehalose, 0.001 g / L to 1.5 g / L L-glutamic acid, 0.00001 g / L to 0.01 g / L L-cysteine, 0.5 g / L to 15 g / L TSB, 0.001 g / L to 1.5 g / L SPS, 0.00001 g / L to 0.01 g / L menaquinone, 0.0001 g / L to 1 g / L pyridoxal hydrochloride, 0.0001 g / L to 1 g / L ferulic acid, 0.1 g / L to 10 g / L sodium hydroxide, 0.001 g / L to 1.0 g / L ascorbic acid, and 0.0001 g / L to 1 g / L hemin chloride.

[0037] 20. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium comprises, in water or in an aqueous solution, about 0.001 g / L to 0.15 g / L iron, 0.0001 g / L to 0.001 g / L copper, 3.0 g / L to 5.0 g / L yeast extract, 0.15 g / L to 1.5 g / L sucrose, 2.5 g / L to 3.5 g / L dextrose, 1.5 g / L to 3.5 g / L D-trehalose, 0.5 g / L to 1 g / L L-glutamic acid, 0.0001 g / L to 0.0012 g / L L-cysteine, 0.5 g / L to 60 g / L TSB, 0.5 g / L to 1.5 g / L SPS, 0.0004 g / L to 0.0012 g / L menaquinone, 0.005 g / L to 0.025 g / L pyridoxal hydrochloride, 0.001 g / L to 0.1 g / L ferulic acid, 0.5 g / L to 3 g / L sodium hydroxide, 0.01 g / L to 0.25 g / L ascorbic acid, and 0.001 g / L to 0.1 g / L hemin chloride.

[0038] 21. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium comprises or consists essentially of about 0.01 g / L of iron, 0.001 g / L of copper, 0.528 g / L of sucrose, 3.2 g / L of dextrose, 3.2 g / L of D-trehalose, 4 g / L of yeast extract, 0.8 g / L of L-glutamic acid, 44 g / L of TSB, 0.8 g / L of SPS, 0.0008 g / L of menaquinone, 0.016 g / L of pyridoxal hydrochloride, 0.008 g / L of ferulic acid, 1.225 g / L of sodium hydroxide, 0.08 g / L of ascorbic acid, 0.0008 g / L of L-cysteine, and 0.008 g / L of hemin chloride.

[0039] 22. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium comprises or consists essentially of about 0.01 g / L of iron, 0.001 g / L of copper, 0.33 g / L of sucrose, 2 g / L of dextrose, 2 g / L of D-trehalose, 2.5 g / L of yeast extract, 0.5 g / L of L-glutamic acid, 27.5 g / L of TSB, 0.5 g / L of SPS, 0.00005 g / L of menaquinone, 0.01 g / L of pyridoxal hydrochloride, 0.005 g / L of ferulic acid, 0.7655 g / L of sodium hydroxide, 0.05 g / L of ascorbic acid, 0.0005 g / L of L-cysteine, and 0.005 g / L of hemin chloride.

[0040] 23. The microbial growth medium according to any one of the foregoing embodiments, wherein the microbial growth medium is capable of supporting the growth of one or more microbial species selected from the following: Abiotrophia defectiva, Acinetobacter lwoffii, Aggregatibacter actinomycetemcomitans, Aerococcus viridins, Alcaligenes faecalis, Bacillus subtilis, Candida albicans, Candida glabrata, Cardiobacterium hominis, Corynebacterium jeikeium, Cryptococcus neoformans, Eikenella corrodens, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Granulicatella adiacens, Haemophilus influenzae, Haemophilus influenzae, Haemophilus influenzae type a, Haemophilus influenzae type b, Haemophilus parainfluenzae, Kingella kingae, Klebsiella pneumoniae, Leuconostoc species, Micrococcus luteus, Neisseria gonorrhoeae, Neisseria meningitidis, Pediococcus acidilactici, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Rothia mucilaginosamucilaginosa), Saccharomyces cerevisiae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguinis.

[0041] 24. The microbial growth medium according to any one of the foregoing embodiments, wherein the growth rate of Candida is increased by, is about, is at least, is at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, or 80%, or is a range defined by any two of the foregoing values.

[0042] 25. The microbial growth medium according to any one of the foregoing embodiments, wherein the TTD of Candida is shortened by, is about, is at least, is at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75%, or 80%, or is a range defined by any two of the foregoing values.

[0043] 26. The microbial growth medium according to any one of the foregoing embodiments, wherein the TTD of Candida is shortened by, is about, is at least, is at least about, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours, or 72 hours, or is a range defined by any two of the foregoing values.

[0044] 27. A culture container for detecting microbial growth, the container comprising:

[0045] The microbial growth medium according to any one of the foregoing embodiments; and

[0046] A sensor for monitoring a parameter of the microbial growth medium, the parameter indicating microbial growth in the microbial growth medium.

[0047] 28. The culture vessel according to embodiment 27, wherein the sensor is separated from the contents of the microbial growth medium by a permeable membrane.

[0048] 29. The culture vessel according to embodiment 27 or 28, wherein the parameter to be monitored is pH, O2, and / or CO2.

[0049] 30. The culture vessel according to any one of embodiments 27 to 29, wherein the sensor includes a pH sensor.

[0050] 31. The culture vessel according to embodiment 30, wherein the pH sensor includes a fluorescent, phosphorescent, or colorimetric pH-responsive reagent.

[0051] 32. The culture vessel according to any one of embodiments 27 to 31, wherein the sensor includes an O2 sensor.

[0052] 33. The culture vessel according to embodiment 32, wherein the pH sensor includes a fluorescent, phosphorescent, or colorimetric O2-responsive reagent.

[0053] 34. The culture vessel according to any one of embodiments 27 to 33, wherein the sensor includes a CO2 sensor.

[0054] 35. The culture vessel according to embodiment 34, wherein the CO2 sensor includes a fluorescent, phosphorescent, or colorimetric pH-responsive reagent.

[0055] 36. The culture vessel according to any one of embodiments 27 to 35, wherein the sensor includes a pH, O2, and / or CO2-sensitive resin.

[0056] 37. The culture vessel according to embodiment 36, wherein the culture vessel contains a pH and / or CO2-sensitive resin in an amount of, about, at least, at least about, not exceeding, or not exceeding about, 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L, 3.0 g / L, 4.0 g / L, 5.0 g / L, 6.0 g / L, 7.0 g / L, 8.0 g / L, 9.0 g / L, or 10.0 g / L, or a range defined by any two of the foregoing values.

[0057] 38. The culture vessel according to any one of embodiments 27 to 37, wherein the vessel further includes a headspace volume containing a gas mixture of O2, CO2, and N2.

[0058] 39. The culture vessel according to embodiment 38, wherein the gas in the headspace comprises:

[0059] about 25% to about 75% O2,

[0060] about 15% to about 45% CO2, and

[0061] about 0% to about 40% N2.

[0062] 40. The culture vessel according to embodiment 38 or 39, wherein the gas in the headspace comprises, consists of, consists essentially of, contains about, contains at least, contains at least about, contains less than or contains less than about, 25%, 30%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, or an O2 within a range defined by any two of the foregoing values.

[0063] 41. The culture vessel according to embodiment 38 or 39, wherein the gas in the headspace comprises about 47% to 60% O2.

[0064] 42. The culture vessel according to embodiment 38 or 39, wherein the gas in the headspace comprises about 52% to 56% O2.

[0065] 43. The culture vessel according to any one of embodiments 38 to 42, wherein the gas in the headspace comprises, consists of, consists essentially of, contains about, contains at least, contains at least about, contains less than or contains less than about, 15%, 20%, 21%, 22%, 23%, 24%, 24.1%, 24.2%, 24.3%, 24.4%, 24.5%, 24.6%, 24.7%, 24.8%, 24.9%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 45%, or a CO2 within a range defined by any two of the foregoing values.

[0066] 44. The culture vessel according to any one of embodiments 38 to 42, wherein the gas in the headspace comprises about 20% to about 30% CO2.

[0067] 45. The culture vessel according to any one of embodiments 38 to 42, wherein the gas in the headspace comprises about 24% to about 25% CO2.

[0068] 46. The culture vessel according to any one of embodiments 36 to 45, wherein the gas in the headspace comprises, comprises about, comprises at least, comprises at least about, comprises less than or comprises less than about, 20%, 25%, 26%, 27%, 28%, 28.1%, 28.3%, 28.3%, 28.4%, 28.5%, 28.6%, 28.7%, 28.8%, 28.9%, 29%, 30%, 31%, 32%, 33%, 34%, 35% or 40%, or N2 in a range defined by any two of the foregoing values.

[0069] 47. The culture vessel according to any one of embodiments 38 to 45, wherein the gas in the headspace comprises about 0% to 30% N2.

[0070] 48. The culture vessel according to any one of embodiments 38 to 45, wherein the gas in the headspace consists of or consists essentially of O2, CO2 and N2.

[0071] 49. The culture vessel according to any one of embodiments 27 to 48, wherein the microbial growth is the growth of a microorganism selected from the following: Abiotrophia defectiva, Acinetobacter lwoffii, Aggregatibacter actinomycetemcomitans, Aerococcus viridans, Alcaligenes faecalis, Bacillus subtilis, Candida albicans, Candida glabrata, Cardiobacterium hominis, Corynebacterium jeikeium, Cryptococcus neoformans, Eikenella corrodens, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Granulicatella adiacens, Haemophilus influenzae, Haemophilus influenzae, Haemophilus influenzae type a, Haemophilus influenzae type b, Haemophilus parainfluenzae, Kingella kingae, Klebsiella pneumoniae, Leuconostoc species, Micrococcus luteus, Neisseria gonorrhoeae, Neisseria meningitidis, Pediococcus acidilactici, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Rothia mucilaginosa, Saccharomyces cerevisiae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus sanguinis.

[0072] 50. A system for detecting the presence of a microorganism in a sample, the system comprising:

[0073] The culture vessel according to any one of embodiments 27 to 49;

[0074] A detector for obtaining a signal from a sensor;

[0075] A computer configured to determine whether the signal obtained by the detector indicates the presence of a microorganism in the microbial growth medium.

[0076] 51. A method for culturing a microorganism in a sample, the method comprising:

[0077] Inoculate the microbial growth medium described in any of the foregoing embodiments with a sample, and culture the microorganisms in the sample in the microbial growth medium.

[0078] 52. The method according to embodiment 51, wherein the method further comprises detecting whether there are microorganisms in the sample.

[0079] 53. The method according to embodiment 51 or 52, wherein the inoculation step comprises adding the sample to the microbial growth medium in the culture container described in any of embodiments 27 to 50.

[0080] 54. The method according to embodiment 53, wherein detecting whether there are the microorganisms in the sample comprises monitoring a signal in the sensor indicating the growth of the microorganisms in the microbial growth medium.

[0081] 55. The method according to embodiment 54, wherein the signal indicates a change in the pH of the microbial growth medium.

[0082] 56. The method according to embodiment 54, wherein the signal indicates a change in CO2 in the gas in the headspace of the culture container.

[0083] 57. The method according to any of embodiments 52 to 55, wherein the microorganism is Candida, and the TTD of Candida is shortened compared with culturing Candida in the same microbial growth medium, wherein the amount of iron in the same microbial growth medium is less than about 0.4 ppm and the amount of copper is less than about 0.04 ppm, or wherein there is no iron and copper in the same microbial growth medium.

[0084] 58. The method according to any of embodiments 51 to 56, wherein the culturing comprises maintaining the microbial medium at a temperature of 35°C to 39°C or 37°C.

[0085] 59. The method according to any of embodiments 51 to 58, wherein the method is performed using the system described in embodiment 50.

[0086] 60. The method according to any of embodiments 51 to 59, wherein the sample is selected from: biological samples, such as blood, serum, plasma, urine, cerebrospinal fluid, pleural effusion, thoracic fluid, thoracentesis fluid, peritoneal fluid, abdominal fluid, ascites, pericardial fluid, bone marrow, synovial fluid; or industrial samples, such as food or pharmaceutical ingredients.

[0087] 61. The culture vessel according to any one of embodiments 51 to 60, wherein the sample is a biological sample from a patient positive for systemic inflammatory response syndrome (SIRS) or a septic patient.

[0088] Brief Description of the Drawings

[0089] Figure 1 An embodiment showing a list of organisms for testing improved TTD in the disclosed microbial growth medium.

[0090] Figure 2 An embodiment showing the TTD results of Wilcoxon paired analysis.

[0091] Figure 3 An embodiment showing the results of TTD testing of Acinetobacter lwoffii in 0 mL, 3 mL, and 10 mL blood samples.

[0092] Figure 4 An embodiment showing the TTD testing of Haemophilus parainfluenzae in 0.5 mL, 3 mL, and 10 mL blood samples.

[0093] Figure 5 An embodiment showing the results of TTD testing of three other strains of Haemophilus parainfluenzae in 0.5 mL, 3 mL, and 10 mL blood samples.

[0094] Figure 6 An embodiment showing the results of TTD testing of Neisseria meningitidis in 0.5 mL, 3 mL, and 10 mL blood samples.

[0095] Figure 7 An embodiment showing the results of TTD testing of Rothia mucilaginosa in 0 mL, 3 mL, and 10 mL blood samples.

[0096] Figure 8 An embodiment showing the results of TTD testing of three other strains of Rothia mucilaginosa in 0 mL, 3 mL, and 10 mL blood samples.

[0097] Figure 9 An embodiment showing the results of TTD testing of Stenotrophomonas maltophilia in 0 mL, 3 mL, and 10 mL blood samples.

[0098] Figure 10 An embodiment showing the results of TTD testing of five other strains of Rothia mucilaginosa in 0 mL, 3 mL, and 10 mL blood samples.

[0099] Figure 11Embodiments showing the results of TTD tests on Streptococcus pneumoniae in 0 mL, 3 mL, and 10 mL blood samples.

[0100] Figure 12 Embodiments showing the results of repeated TTD tests on Streptococcus pneumoniae in 0 mL, 3 mL, and 10 mL blood samples.

[0101] Figure 13 Embodiments showing the list of organisms for percent recovery testing.

[0102] Figure 14 Embodiments showing the results of percent recovery testing using McNemar’s Chi Square test in BD Plus Aerobic / 26F BACTEC (PFS) compared to the disclosed microbial growth medium (SJ).

[0103] Figure 15 Embodiments showing false positive test results.

[0104] Figure 16 Embodiments showing the list of organisms for DVE (delayed vial entry) testing.

[0105] Figure 17 Embodiments showing the comparison of DVE testing in BD Plus Aerobic / 26F BACTEC (PFS) with DVE testing in the disclosed microbial growth medium (SJ).

[0106] Figure 18 Embodiments showing a table of organisms, antibiotics, and antibiotic concentrations for growth support testing.

[0107] Figure 19 Embodiments showing the results of growth support testing using McNemar’s Chi Square test in BD Plus Aerobic / 26F BACTEC (PFS) compared to the disclosed microbial growth medium (SJ).

[0108] Figure 20 Embodiments showing a table of GPC, GNB, yeast, Neisseria / Haemophilus, and GP / GNCB groups of microorganisms and representative microorganisms belonging to various categories.

[0109] Figure 21 Embodiments showing a table of combinations of iron concentration and O2 concentration for improved TTD testing.

[0110] Figure 22Embodiments showing histograms and quantification of the TTD of microorganisms in blood volumes of 0 mL, 0.5 mL, 3 mL or 10 mL.

[0111] Figure 23 Embodiments showing histograms of the TTD of the GC / Haem, GNB, GPB / GNCB, GPC and yeast groups in microbial growth medium bottles containing 47%, 54% and 60% O2 in the gas of the headspace.

[0112] Figure 24 Embodiments showing histograms of the TTD of the GC / Haem, GNB, GPB / GNCB, GPC and yeast groups in microbial growth media containing 0 g / L, 0.01 g / L, 0.035 g / L and 0.06 g / L iron.

[0113] Figure 25 Embodiments showing dot plots of the TTD of the GC / Haem, GNB, GPB / GNCB, GPC and yeast groups in microbial growth media containing 0 g / L, 0.01 g / L, 0.035 g / L and 0.06 g / L iron.

[0114] Figure 26 Embodiments showing dot plots of the TTD of the GC / Haem, GNB, GPB / GNCB, GPC and yeast groups in microbial growth media containing 47%, 54% and 60% O2 in the gas of the headspace.

[0115] Figure 27 Embodiments showing dot plots of the TTD of various microorganisms in microbial growth media containing 0 g / L, 0.01 g / L, 0.035 g / L and 0.06 g / L iron.

[0116] Figure 28 Embodiments showing dot plots of the TTD of various microorganisms in microbial growth medium bottles containing 47%, 54% and 60% O2 in the gas of the headspace.

[0117] Figure 29 Embodiments showing histograms of the TTD of yeast in microbial growth media containing 0 g / L, 0.01 g / L, 0.035 g / L and 0.06 g / L iron.

[0118] Figure 30Shows embodiments of histograms of the combined TTDs of the GPC, GNB, Neisseria / Haemophilus, and GP / GNCB groups of microorganisms in microbial growth media containing 0 g / L, 0.01 g / L, 0.035 g / L, and 0.06 g / L iron.

[0119] Figure 31 Shows embodiments of histograms showing improved TTD in yeast compared to the TTDs of the GPC, GNB, Neisseria / Haemophilus, and GP / GNCB groups of microorganisms.

[0120] Figure 32 Shows embodiments of bivariate fits of the microbial TTD in the disclosed microbial growth media bottles (ICE control standard) with a gas having a headspace containing 47% O2.

[0121] Figure 33 Shows embodiments of summary histograms and Wilcoxon paired analysis data of the microbial TTD in the disclosed microbial growth media bottles (ICE control standard) with a gas having a headspace containing 47% O2.

[0122] Figure 34 Shows embodiments of bivariate fits of the microbial TTD in the disclosed microbial growth media bottles (ICE control 54) with a gas having a headspace containing 54% O2.

[0123] Figure 35 Shows embodiments of summary histograms and Wilcoxon paired analysis data of the microbial TTD in the disclosed microbial growth media bottles (ICE control 54) with a gas having a headspace containing 54% O2.

[0124] Figure 36 Shows embodiments of bivariate fits of the microbial TTD in the disclosed microbial growth media bottles (ICE control 60) with a gas having a headspace containing 54% O2.

[0125] Figure 37 Shows embodiments of summary histograms and Wilcoxon paired analysis data of the microbial TTD in the disclosed microbial growth media bottles (ICE control 54) with a gas having a headspace containing 60% O2.

[0126] Figure 38 Shows embodiments of bivariate fits of the microbial TTD in the disclosed microbial growth media bottles containing a microbial growth medium with 0.01 g / L iron and a gas headspace containing 47% O2 (ICE 0.01 Fe standard).

[0127] Figure 39 Shows embodiments of a summary histogram of microbial TTD and Wilcoxon paired analysis data in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.01 g / L iron and a gas headspace with 47% O2 (ICE 0.01 Fe standard).

[0128] Figure 40 Shows embodiments of a bivariate fit of microbial TTD in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.035 g / L iron and a gas headspace with 47% O2 (ICE 0.035 Fe standard).

[0129] Figure 41 Shows embodiments of a summary histogram of microbial TTD and Wilcoxon paired analysis data in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.035 g / L iron and a gas headspace with 47% O2 (ICE 0.035 Fe standard).

[0130] Figure 42 Shows embodiments of a bivariate fit of microbial TTD in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.06 g / L iron and a gas headspace with 47% O2 (ICE 0.06 Fe standard).

[0131] Figure 43 Shows embodiments of a summary histogram of microbial TTD and Wilcoxon paired analysis data in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.06 g / L iron and a gas headspace with 47% O2 (ICE 0.06 Fe standard).

[0132] Figure 44 Shows embodiments of a bivariate fit of microbial TTD in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.01 g / L iron and a gas headspace with 54% O2 (ICE 0.01 Fe 54).

[0133] Figure 45Shows an embodiment of the summary histogram and Wilcoxon paired analysis data of the microbial TTD in the disclosed microbial growth medium bottle, which contains a microbial growth medium containing 0.01 g / L iron and a gas headspace containing 54% O2 (ICE 0.01 Fe 54).

[0134] Figure 46 Shows an embodiment of the bivariate fit of the microbial TTD in the disclosed microbial growth medium bottle, which contains a microbial growth medium containing 0.35 g / L iron and a gas headspace containing 54% O2 (ICE 0.035 Fe 54).

[0135] Figure 47 Shows an embodiment of the summary histogram and Wilcoxon paired analysis data of the microbial TTD in the disclosed microbial growth medium bottle, which contains a microbial growth medium containing 0.035 g / L iron and a gas headspace containing 54% O2 (ICE 0.035 Fe 54).

[0136] Figure 48 Shows an embodiment of the bivariate fit of the microbial TTD in the disclosed microbial growth medium bottle, which contains a microbial growth medium containing 0.06 g / L iron and a gas headspace containing 54% O2 (ICE 0.06 Fe 54).

[0137] Figure 49 Shows an embodiment of the summary histogram and Wilcoxon paired analysis data of the microbial TTD in the disclosed microbial growth medium bottle, which contains a microbial growth medium containing 0.06 g / L iron and a gas headspace containing 54% O2 (ICE 0.06 Fe 54).

[0138] Figure 50 Shows an embodiment of the bivariate fit of the microbial TTD in the disclosed microbial growth medium bottle, which contains a microbial growth medium containing 0.01 g / L iron and a gas headspace containing 60% O2 (ICE 0.01 Fe 60).

[0139] Figure 51Shows an embodiment of a summary histogram and Wilcoxon paired analysis data of microbial TTD in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.01 g / L iron and a gas headspace with 60% O2 (ICE 0.01 Fe 60).

[0140] Figure 52 Shows a representative bivariate fit of microbial TTD in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial medium with 0.035 g / L iron and a gas headspace with 60% O2 (ICE 0.035 Fe 60).

[0141] Figure 53 Shows an embodiment of a summary histogram and Wilcoxon paired analysis data of microbial TTD in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.035 g / L iron and a gas headspace with 60% O2 (ICE 0.035 Fe 60).

[0142] Figure 54 Shows an embodiment of a bivariate fit of microbial TTD in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.06 g / L iron and a gas headspace with 60% O2 (ICE 0.06 Fe 60).

[0143] Figure 55 Shows an embodiment of a summary histogram and Wilcoxon paired analysis data of microbial TTD in the disclosed microbial growth medium bottles, the microbial growth medium bottles containing a microbial growth medium with 0.06 g / L iron and a gas headspace with 60% O2 (ICE 0.06 Fe 60).

[0144] Figure 56 Shows an embodiment of the ANOVA results of the effects of different concentrations of iron and O2 on microbial TTD in a microbial growth medium.

[0145] Figure 57 Shows an embodiment of the ANOVA results of the parameter estimates and prediction equations of TTD for the GC / Haem, GNB, GPB / GNCB, GPC, and yeast groups of microorganisms in a microbial growth medium. Detailed Description

[0146] Although certain embodiments and examples are disclosed below, the subject matter of the present invention extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses and to their modifications and equivalents. Accordingly, the scope of the presently disclosed invention is not limited by any particular embodiment described below. For example, in any of the methods or processes disclosed herein, the acts or operations of the method or process may be performed in any suitable order and need not be limited to any particular disclosed order. The various operations may be described as multiple discrete operations in turn, in a manner that may be helpful in understanding some embodiments; however, the order of the description should not be construed as implying that these operations are order-dependent. Additionally, the structures, systems, and / or devices described herein may be embodied as integrated components or separate components. For purposes of comparing various embodiments, certain aspects and advantages of these embodiments are described. Any such aspects and advantages need not be achieved by any particular embodiment. Thus, for example, the various embodiments may be implemented in a manner that achieves or optimizes one advantage or a group of advantages as taught herein, without necessarily achieving other aspects or advantages as may also be taught or suggested herein.

[0147] Early detection of bacterial and fungal infections is crucial for guiding antimicrobial therapy in septic patients. Sepsis is a potentially life-threatening condition that occurs when the body's response to infection damages its own tissues. Sepsis can progress to septic shock, which is a sharp drop in the patient's blood pressure that can lead to severe organ damage and death. As of 2013, there were approximately 20,000 sepsis-related deaths worldwide each day. This amounts to an estimated 18,000,000 deaths per year. Between 2000 and 2008, the number of hospital admissions for sepsis in the United States more than doubled. In 2009, sepsis was the most expensive cause of hospitalization in the United States, with total hospital costs approaching $15.4 billion. This increased cost of hospitalization provided little benefit to septic patients, as in 2009, the mortality rate in the United States was eight times higher than the mortality rate from other hospitalizations. Each hour of delay in administering effective antimicrobials increases the mortality rate by 7.6%. Microbiological growth medium testing allows for the identification of microorganisms in subjects with systemic infections. In addition to other nutrients, existing microbiological growth media contain an unspecified amount of iron and copper and typically have a long time to detection (TTD) of microorganisms in biological samples.

[0148] In some embodiments, the present disclosure relates to microbiological growth media having an improved microbial growth rate and / or microbial TTD. In some embodiments, the microorganism is Candida Candida)。In some embodiments, the microorganism is Candida albicans or Candida glabrata. In some embodiments, the microorganism is in a biological sample. In some embodiments, the biological sample is blood. In some embodiments, the biological sample is serum or plasma. In some embodiments, the biological sample is urine. In some embodiments, the biological sample is a body fluid that is sterile in a healthy patient, such as blood, urine, cerebrospinal fluid, pleural effusion, thoracic fluid, thoracentesis fluid, peritoneal fluid, ascites, peritoneal effusion, pericardial fluid, bone marrow, synovial fluid. In some embodiments, the biological sample is obtained from a septic subject. In some embodiments, the biological sample is obtained from a subject positive for systemic inflammatory response syndrome (SIRS). In some embodiments, the subject has a systemic infection. In some embodiments, the systemic infection is a bacterial infection. In some embodiments, the bacterium is a Gram-negative bacterium. In some embodiments, the bacterium is a Gram-positive bacterium. In some embodiments, the infection is a yeast infection. In some embodiments, the infection is an A defectus, Acinetobacter lwoffii, Aggregatibacter actinomycetemcomitans, Aerococcus viridans, Alcaligenes faecalis, Bacillus subtilis, Candida albicans, Candida glabrata, Cardiobacterium hominis, Corynebacterium jeikeium, Cryptococcus neoformans, Eikenella corrodens, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Granulicatella adiacens, Haemophilus influenzae, Haemophilus influenzae, Haemophilus influenzae type a, Haemophilus influenzae type b, Haemophilus parainfluenzae, Kingella kingae, Klebsiella pneumoniae, Leuconostoc species, Micrococcus luteus, Neisseria gonorrhoeae, Neisseria meningitidis, Pediococcus acidilactici, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Rothia mucilaginosa, Saccharomyces cerevisiae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus sanguinis infection. In some embodiments, the sample is from an industrial application in which the sample is tested for sterility, such as a sample of a food or drug product (e.g., for USP applications).

[0149] In some embodiments, the microbial growth medium contains iron. In some embodiments, the iron is present as ferric iron (Fe(III) or Fe 3+ )). In some embodiments, the iron is present as ferrous iron (Fe(II) or Fe 2+) Provided. In some embodiments, the iron comprises an iron salt or an iron complex. In some embodiments, the iron is provided as ammonium ferric citrate, ferric chloride, ferric sulfate, ferric nitrate, and / or other suitable ferric salts or complexes. In some embodiments, the iron is provided as the corresponding ferrous salts or complexes described above. In some embodiments, the amount of iron is, is about, at least, at least about, not more than, or not more than about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 28 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 170 μM, 175 μM, 200 μM, 225 μM, 250 μM, 275 μM, 300 μM, 325 μM, 350 μM, 375 μM, or 400 μM, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of iron is or is about: 1 μM to 400 μM, 1 μM to 350 μM, 1 μM to 200 μM, 1 μM to 100 μM, 1 μM to 50 μM, 2 μM to 400 μM, 2 μM to 350 μM, 25 μM to 400 μM, 25 μM to 325 μM, 25 μM to 200 μM, 25 μM to 100 μM, or 25 μM to 75 μM. In some embodiments, the amount of iron is, is about, at least, at least about, not more than, or not more than about 28 μM, 100 μM, or 170 μM. In some embodiments, the amount of iron is or is about 28 μM to 170 μM. In some embodiments, the amount of iron is, is about, at least, at least about, not more than, or not more than about 1 ppm, 1.5 ppm, 2 ppm, 2.5 ppm, 3 ppm, 3.5 ppm, 4 ppm, 4.5 ppm, 5 ppm, 5.5 ppm, 6 ppm, 6.5 ppm, 7 ppm, 7.5 ppm, 8 ppm, 8.5 ppm, 9 ppm, 9.5 ppm, or 10 ppm, or within a range defined by any two of the foregoing values. For example, in some embodiments, the amount of iron is or is about: 1 ppm to 10 ppm, 1 ppm to 7.5 ppm, 1 ppm to 5 ppm, 2.5 ppm to 10 ppm, 2.5 ppm to 7.5 ppm, 2.5 ppm to 5 ppm, 5 ppm to 10 ppm, or 5 to 7.5 ppm.

[0150] In some embodiments, the microbial growth medium comprises copper. In some embodiments, the copper is in the divalent form (Cu(II) or Cu 2+)Present. In some embodiments, copper includes a copper salt or a copper complex. In some embodiments, copper is provided as copper sulfate, copper chloride, copper nitrate, copper bromide, copper chlorate, copper(II) gluconate, or other suitable copper salts or complexes. In some embodiments, the amount of copper is, is about, at least, at least about, not more than, or not more than about 0 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 650 μM, or 700 μM of copper, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of copper is or is about: 0 μM to 700 μM, 0 μM to 650 μM, 0 μM to 500 μM, 0 μM to 300 μM, 0 μM to 100 μM, 0 μM to 50 μM, 0 μM to 25 μM, 0.1 μM to 100 μM, 0.1 μM to 50 μM, 0.1 μM to 25 μM, 0.5 μM to 650 μM, 0.5 μM to 300 μM, 0.5 μM to 100 μM, 0.5 μM to 50 μM, or 0.5 μM to 25 μM. In some embodiments, the amount of copper is, is about, at least, at least about, not more than, or not more than about 0.05 ppm, 0.075 ppm, 0.1 ppm, 0.2 ppm, 0.3 ppm, 0.4 ppm, or 0.5 ppm, or within a range defined by any two of the foregoing values. For example, in some embodiments, the amount of iron is or is about: 0.05 ppm to 0.5 ppm, 0.05 ppm to 0.3 ppm, 0.05 ppm to 2 ppm, 0.1 ppm to 0.5 ppm, or 0.1 ppm to 0.3 ppm.

[0151] In some embodiments, the enhanced microbial growth medium comprises a yeast nitrogen base. In some embodiments, the yeast nitrogen base is lacking in amino acids. In some embodiments, the yeast nitrogen base is lacking in histidine, methionine, tryptophan, and / or any combination thereof. In some embodiments, the yeast nitrogen base is lacking in amino acids other than histidine, methionine, and / or tryptophan. In some embodiments, the yeast nitrogen base with or without certain amino acids is omitted from or not present in the microbial growth medium. In some embodiments, omitting the yeast nitrogen base from the microbial growth medium minimizes precipitation of iron from solution. In some embodiments, the yeast nitrogen base is present in an amount less than the amount that causes precipitation of iron from solution in the microbial growth medium. In some embodiments, the amount of the yeast nitrogen base is, is about, at least, at least about, not more than, or not more than about 0 g / L, 0.01 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1.0 g / L of yeast nitrogen base, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of the yeast nitrogen base is or is about 0.00 g / L to 1 g / L, 0.00 g / L to 0.8 g / L, 0.00 g / L to 0.4 g / L, 0.00 g / L to 0.1 g / L, 0.1 g / L to 1 g / L, 0.1 g / L to 0.8 g / L, or 0.1 g / L to 0.4 g / L of yeast nitrogen base.

[0152] In some embodiments, the microbial growth medium comprises yeast extract. In some embodiments, the amount of the yeast extract is, is about, at least, at least about, not more than, or not more than about 0.0 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, 3.0 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L, 3.8 g / L, 3.9 g / L, 4.0 g / L, 4.1 g / L, 4.2 g / L, 4.3 g / L, 4.4 g / L, 4.5 g / L, 5.0 g / L, 6.0 g / L, 7.0 g / L, 8.0 g / L, 9.0 g / L, or 10.0 g / L of yeast extract, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of the yeast extract is or is about 1.0 g / L to 10.0 g / L, 1.0 g / L to 4.0 g / L, 2.5 g / L to 10.0 g / L, or 2.5 g / L to 4.0 g / L.

[0153] In some embodiments, the microbial growth medium comprises one or more carbohydrates. In some embodiments, the carbohydrate comprises sucrose. In some embodiments, the carbohydrate comprises glucose, fructose, or another suitable carbohydrate. In some embodiments, the amount of sucrose is, is about, at least, at least about, not more than, or not more than about 0.15 g / L, 0.25 g / L, 0.30 g / L, 0.31 g / L, 0.32 g / L, 0.33 g / L, 0.34 g / L, 0.35 g / L, 0.40 g / L, 0.45 g / L, 0.46 g / L, 0.47 g / L, 0.48 g / L, 0.49 g / L, 0.50 g / L, 0.51 g / L, 0.52 g / L, 0.53 g / L, 0.54 g / L, 0.55 g / L, 0.6 g / L, 0.75 g / L, or 1.0 g / L of sucrose, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of sucrose is or is about 0.15 g / L to 1.0 g / L, 0.30 g / L to 1.0 g / L, or 0.30 g / L to 0.60 g / L. In some embodiments, the carbohydrate comprises dextrose. In some embodiments, the microbial growth medium comprises or comprises about 1.0 g / L to 10.0 g / L of dextrose. In some embodiments, the amount of dextrose is, is about, at least, at least about, not more than, or not more than about 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, 2.6 g / L, 2.6 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L, 4.0 g / L, 4.5 g / L, 5.0 g / L, 6.0 g / L, 7.0 g / L, 8.0 g / L, 9.0 g / L, or 10.0 g / L of dextrose, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of dextrose is 1.0 g / L to 10.0 g / L, 1.0 g / L to 5.0 g / L, 1.0 g / L to 3.2 g / L, 2.0 g / L to 10.0 g / L, or 2.0 g / L to 3.2 g / L. In some embodiments, the microbial growth medium comprises both sucrose and dextrose. In some embodiments, the microbial growth medium comprises sucrose or dextrose. In some embodiments, the carbohydrate comprises D-trehalose.In some embodiments, the amount of D-trehalose is, is about, is at least, is at least about, does not exceed or does not exceed about 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, 2.6 g / L, 2.6 g / L, 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L, 4.0 g / L, 4.5 g / L, 5.0 g / L, 6.0 g / L, 7.0 g / L, 8.0 g / L, 9.0 g / L or 10.0 g / L of D-trehalose, or is a range defined by any two of the foregoing values. For example, in some embodiments, the amount of D-trehalose is or is about 1.0 g / L to 10.0 g / L, 1.0 g / L to 5.0 g / L, 1.0 g / L to 3.2 g / L, 2.0 g / L to 10.0 g / L or 2.0 g / L to 3.2 g / L. In some embodiments, the microbial growth medium comprises sucrose, dextrose and / or D-trehalose. In some embodiments, another suitable carbohydrate (e.g., glucose) replaces sucrose, dextrose and / or D-trehalose. In some embodiments, the concentration of the carbohydrate in the microbial growth medium is specifically selected for its suitability to support the growth of one or more microorganisms.

[0154] In some embodiments, the microbial growth medium comprises one or more amino acids. In some embodiments, the amino acids are isoleucine, leucine, valine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, citrulline, GABA, hydroxyproline, oxoproline, ornithine, asparagine, aspartic acid, aspartate, alanine, arginine, cysteine, L-cysteine, cystine, glutamine, glutamic acid, L-glutamic acid, glutamate, glycine, proline, serine, tyrosine, acetylhydroxyproline, alanyl-glutamine, and / or glutathione. In some embodiments, the amino acid is L-glutamic acid. In some embodiments, the amount of L-glutamic acid is, is about, at least, at least about, not more than, or not more than about 0.25 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, or 1.5 g / L of L-glutamic acid, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of L-glutamic acid is or is about 0.25 g / L to 1.5 g / L, 0.25 g / L to 1.0 g / L, 0.25 g / L to 0.8 g / L, 0.25 g / L to 0.5 g / L, 0.5 g / L to 1.5 g / L, or 0.5 g / L to 0.8 g / L. In some embodiments, the amino acid is L-cysteine. In some embodiments, the amount of L-cysteine is, is about, at least, at least about, not more than, or not more than about 0.0001 g / L, 0.0002 g / L, 0.0003 g / L, 0.0004 g / L, 0.0005 g / L, 0.0006 g / L, 0.007 g / L, 0.0008 g / L, 0.0009, or 0.001 g / L of L-cysteine, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of L-cysteine is or is about 0.0001 g / L to 0.001 g / L, 0.0001 g / L to 0.0008 g / L, 0.0005 g / L to 0.001 g / L, or 0.0005 g / L to 0.0008 g / L. In some embodiments, the microbial growth medium comprises both L-glutamic acid and L-cysteine. In some embodiments, the amino acids and / or the concentration of the amino acids in the microbial growth medium are selected for their suitability in promoting the growth of one or more designated microorganisms.

[0155] In some embodiments, the microbial growth medium comprises tryptic soy broth (TSB). In some embodiments, the amount of TSB is, is about, at least, at least about, not more than, or not more than about 15 g / L, 20 g / L, 25 g / L, 27.5 g / L, 30 g / L, 35 g / L, 40 g / L, 44 g / L, 45 g / L, 50 g / L, 55 g / L, or 60 g / L of TSB, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of TSB is or is about 15 g / L to 60 g / L, 15 g / L to 44 g / L, 15 g / L to 27.5 g / L, 27.5 g / L to 60 g / L, or 27.5 g / L to 44 g / L.

[0156] In some embodiments, the microbial growth medium comprises sodium polyanethol sulfonate (SPS). In some embodiments, the amount of SPS is, is about, at least, at least about, not more than, or not more than about 0.25 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, or 1.5 g / L of SPS, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of SPS is or is about 0.25 g / L to 1.5 g / L, 0.25 g / L to 1.0 g / L, 0.25 g / L to 0.8 g / L, 0.5 g / L to 1.5 g / L, or 0.5 g / L to 0.8 g / L.

[0157] In some embodiments, the microbial growth medium comprises menaquinone. In some embodiments, the amount of menaquinone is, is about, at least, at least about, not more than, or not more than about 0.001 g / L, 0.002 g / L, 0.003 g / L, 0.004 g / L, 0.005 g / L, 0.006 g / L, 0.007 g / L, 0.008 g / L, 0.009 g / L, or 0.01 g / L of menaquinone, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of menaquinone is or is about 0.001 g / L to 0.01 g / L, 0.001 g / L to 0.008 g / L, 0.005 g / L to 0.01 g / L, or 0.005 g / L to 0.008 g / L.

[0158] In some embodiments, the microbial growth medium comprises pyridoxal hydrochloride. In some embodiments, the amount of pyridoxal hydrochloride is, is about, at least, at least about, not more than, or not more than about 0.005 g / L, 0.010 g / L, 0.011 g / L, 0.012 g / L, 0.013 g / L, 0.014 g / L, 0.015 g / L, 0.016 g / L, 0.017 g / L, 0.018 g / L, 0.019 g / L, 0.020 g / L, 0.025 g / L, or 0.0301 g / L of pyridoxal hydrochloride, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of pyridoxal hydrochloride is or is about 0.005 g / L to 0.03 g / L, 0.01 g / L to 0.03 g / L, 0.01 g / L to 0.02 g / L, or 0.01 g / L to 0.016 g / L.

[0159] In some embodiments, the microbial growth medium comprises ferulic acid. In some embodiments, the amount of ferulic acid is, is about, at least, at least about, not more than, or not more than about 0.010 g / L, 0.020 g / L, 0.030 g / L, 0.040 g / L, 0.050 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, or 0.15 g / L of ferulic acid, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of ferulic acid is or is about 0.010 g / L to 0.15 g / L, 0.01 g / L to 0.1 g / L, 0.01 g / L to 0.08 g / L, 0.05 g / L to 0.15 g / L, or 0.05 g / L to 0.08 g / L.

[0160] In some embodiments, the microbial growth medium comprises sodium hydroxide. In some embodiments, the amount of sodium hydroxide is, is about, at least, at least about, not more than, or not more than about 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, or 1.5 g / L of sodium hydroxide, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of sodium hydroxide is or is about 0.5 g / L to 1.5 g / L, 0.5 g / L to 1.225 g / L, 0.5 g / L to 1.0 g / L, 0.75 g / L to 1.5 g / L, or 0.75 g / L to 1.225 g / L of sodium hydroxide.

[0161] In some embodiments, the microbial growth medium comprises an antioxidant. In some embodiments, the antioxidant comprises ascorbic acid. In some embodiments, the amount of ascorbic acid is, is about, at least, at least about, not more than, or not more than about 0.025 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.10 g / L, or 0.15 g / L ascorbic acid, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of ascorbic acid is or is about 0.025 g / L to 0.15 g / L, 0.025 g / L to 0.10 g / L, 0.025 g / L to 0.08 g / L, 0.05 g / L to 0.15 g / L, 0.05 g / L to 0.10 g / L, or 0.05 g / L to 0.08 g / L.

[0162] In some embodiments, the microbial growth medium comprises hemin. In some embodiments, the amount of hemin is, is about, at least, at least about, not more than, or not more than about 0.010 g / L, 0.020 g / L, 0.030 g / L, 0.040 g / L, 0.050 g / L, 0.06 g / L, 0.07 g / L, 0.08 g / L, 0.09 g / L, 0.1 g / L, or 0.15 g / L hemin, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of hemin is or is about 0.010 g / L to 0.15 g / L, 0.01 g / L to 0.1 g / L, 0.01 g / L to 0.08 g / L, 0.05 g / L to 0.15 g / L, or 0.05 g / L to 0.08 g / L.

[0163] In some embodiments, the microbial growth medium comprises water (e.g., deionized (DI) water), iron, copper, sucrose, dextrose, D-trehalose, yeast extract, L-glutamic acid, tryptic soy broth (TSB), sodium polyanetholesulfonate (SPS), menaquinone, pyridoxal hydrochloride, ferulic acid, sodium hydroxide, ascorbic acid, L-cysteine, and hemin. In some embodiments, the microbial growth medium comprises or comprises from about 0.001 g / L to 0.15 g / L iron, such as ferric iron (e.g., provided as ammonium ferric citrate); from 0.0001 g / L to 0.0020 g / L copper, such as cupric copper (e.g., provided as copper sulfate); from 1.0 g / L to 10.0 g / L yeast extract; from 0.15 g / L to 1.0 g / L sucrose; from 1.0 g / L to 10.0 g / L dextrose; from 1.0 g / L to 10.0 g / L D-trehalose; from 0.001 g / L to 0.01 g / L L-glutamic acid; from 0.0001 g / L to 0.001 g / L L-cysteine; from 15 g / L to 60 g / L TSB; from 0.25 g / L to 1.5 g / L SPS; from 0.001 g / L to 0.01 g / L menaquinone; from 0.005 g / L to 0.030 g / L pyridoxal hydrochloride; from 0.010 g / L to 0.15 g / L ferulic acid; from 0.5 g / L to 1.5 g / L sodium hydroxide; from 0.025 g / L to 0.15 g / L ascorbic acid; from 0.01 g / L to 0.15 g / L hemin. In some embodiments, the microbial growth medium comprises water (e.g., DI water); from 0.01 g / L to 0.06 g / L iron, such as ferric iron (e.g., provided as ammonium ferric citrate); from 0.0005 g / L to 0.0015 g / L copper, such as cupric copper (e.g., provided as copper sulfate); from 2.5 g / L to 4.0 g / L yeast extract; from 0.35 g / L to 0.6 g / L sucrose; from 2.0 g / L to 3.2 g / L dextrose; from 2.0 g / L to 3.2 g / L D-trehalose; from 0.5 g / L to 0.8 g / L L-glutamic acid; from 0.0005 g / L to 0.0008 g / L L-cysteine; from 2.5 g / L to 44 g / L TSB; from 0.5 g / L to 0.8 g / L SPS; from 0.005 g / L to 0.008 g / L menaquinone; from 0.010 g / L to 0.016 g / L pyridoxal hydrochloride; from 0.05 g / L to 0.08 g / L ferulic acid; from 0.75 g / L to 1.225 g / L sodium hydroxide; from 0.05 g / L to 0.08 g / L ascorbic acid; from 0.05 g / L to 0.08 g / L hemin.In some embodiments, the microbial growth medium comprises or consists of about 0.01 g / L iron, 0.001 g / L copper, 0.528 g / L sucrose, 3.2 g / L dextrose, 3.2 g / L D-trehalose, 4 g / L yeast extract, 0.8 g / L L-glutamic acid, 44 g / L TSB, 0.8 g / L SPS, 0.0008 g / L menaquinone, 0.016 g / L pyridoxal hydrochloride, 0.008 g / L ferulic acid, 1.225 g / L sodium hydroxide, 0.08 g / L ascorbic acid, 0.0008 g / L L-cysteine, and 0.008 g / L hemin. In some embodiments, the bacterial growth medium comprises or consists of about 0.01 g / L iron, 0.001 g / L copper, 0.33 g / L sucrose, 2 g / L dextrose, 2 g / L D-trehalose, 2.5 g / L yeast extract, 0.5 g / L L-glutamic acid, 27.5 g / L TSB, 0.5 g / L SPS, 0.00005 g / L menaquinone, 0.01 g / L pyridoxal hydrochloride, 0.005 g / L ferulic acid, 0.7655 g / L sodium hydroxide, 0.05 g / L ascorbic acid, 0.0005 g / L L-cysteine, and 0.005 g / L hemin.

[0164] In some embodiments, the present disclosure relates to a microbial growth medium having an improved growth rate and / or TTD for Candida (e.g., Candida albicans or Candida glabrata), other yeasts, and / or other microorganisms. In some embodiments, the microorganism is in a biological sample. In some embodiments, the biological sample is a blood, serum, plasma, or urine sample. In some embodiments, the biological sample is obtained from a septic subject. In some embodiments, the biological sample is obtained from a systemic inflammatory response syndrome (SIRS)-positive subject. In some embodiments, adding iron and / or copper to the microbial growth medium as disclosed herein shortens the TTD of Candida (e.g., Candida albicans or Candida glabrata), other yeasts, and / or other microorganisms in the biological sample added to the growth medium. In some embodiments, adding iron and / or copper to the microbial growth medium as disclosed herein shortens the TTD of Candida (e.g., Candida albicans or Candida glabrata), other yeasts, and / or other microorganisms in the blood, serum, plasma, or urine sample added to the microbial growth medium. In some embodiments, the microbial growth medium has a similar percent recovery, delay to vial entry (DVE), false positive rate, and antimicrobial growth support as the same microbial growth medium without the iron and copper contents disclosed herein. In some embodiments, the microbial growth medium has an enhanced percent recovery, DVE, false positive rate, antimicrobial growth support, or any combination thereof as compared to the same growth medium without the iron and copper contents disclosed herein. In some embodiments, adding iron and / or copper to the microbial growth medium as disclosed herein increases the growth rate of Candida (e.g., Candida albicans or Candida glabrata), other yeasts, and / or other microorganisms. In some embodiments, the microbial growth medium has, has about, has at least, has at least about, has no more than, or has no more than about a 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 75%, or 80% increase in the growth rate of the microorganism (e.g., Candida, e.g., Candida albicans or Candida glabrata) in the biological sample, or the growth rate increase is in a range defined by any two of the foregoing values. For example, in some embodiments, the increased microbial growth rate is or is about a 10% to 80%, 10% to 50%, 10% to 20%, 25% to 80%, or 25% to 50% increase. In some embodiments, the microbial growth medium has, has about, has at least, has at least about, has no more than, or has no more than about a 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 60%, 70%, 75%, or 80% shortening of the TTD of the microorganism (e.g., Candida, e.g., Candida albicans or Candida glabrata) in the biological sample, or the TTD shortening is in a range defined by any two of the foregoing values.For example, in some embodiments, the TTD is reduced to or is about 10% to 80%, 10% to 50%, 10% to 20%, 25% to 80%, or 25% to 50% reduction. In some embodiments, the TTD of the microorganisms in the microbial growth medium is reduced to, is about, is at least, is at least about, is not more than, or is not more than about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours, or 72 hours, or the TTD is reduced to a range defined by any two of the foregoing values. For example, in some embodiments, the TTD is reduced to or is about 0 to 72 hours.

[0165] In some embodiments, the reduced TTD of Candida (e.g., Candida, such as Candida albicans or Candida glabrata) cultured in a microbial growth medium is determined by: modifying a culture container (e.g., a BDBACTEC blood culture bottle, Becton, Dickinson and Co.) that contains 30 ml of a microbial growth medium with and without iron and / or copper and 0.5 mL, 3 mL, 10 mL of blood (or an optional alternative biological sample (e.g., serum, saliva, or urine)); and inoculating 0.1 mL of Candida with a final target concentration of 10 to 100 CFU per bottle, such as 50 CFU. The Candida is then cultured at about 30°C to 37°C for up to about 120 hours. A signal (e.g., fluorescence) indicating the presence of Candida (e.g., a change in pH and / or CO2) in the culture container is monitored, and the TTD of Candida in the microbial growth medium with and without iron and / or copper is determined. In some embodiments, the median of the receipt intervals of the signals indicating the presence of Candida in paired culture containers is compared. In some embodiments, culturing Candida, detecting the presence of Candida, and determining the TTD are performed on an automated instrument, such as a BACTEC FX instrument (Becton, Dickinson and Co.). In some embodiments, paired bottles with and without iron and / or copper enter adjacent positions on the instrument simultaneously.

[0166] In some embodiments, culture containers for detecting microbial growth are provided. In some embodiments, the microbial organisms include Candida, such as Candida albicans or Candida glabrata, other yeasts, and / or other microorganisms. In some embodiments, the culture container contains a microbial growth medium of any of the embodiments described herein. In some embodiments, the culture container includes a sensor for monitoring a parameter of the microbial growth medium (e.g., providing a signal proportional thereto) indicative of the presence of microbial growth in the microbial growth medium. In some embodiments, the sensor is a pH sensor. In some embodiments, the sensor is a CO2 sensor. In some embodiments, the sensor is a dissolved CO2 sensor. In some embodiments, the sensor is an O2 sensor. In some embodiments, the sensor is a dye. In some embodiments, the dye is a pH-sensitive dye. In some embodiments, the dye is an O2-sensitive dye. In some embodiments, the sensor is a CO2 or dissolved CO2-sensitive dye. In some embodiments, the pH, O2, CO2, or dissolved CO2 sensor is a pH, O2, CO2, or dissolved CO2-sensitive resin. In some embodiments, the culture container has, has about, has at least, has at least about, has no more than, or has no more than about 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L, 3.0 g / L, 4.0 g / L, 5.0 g / L, 6.0 g / L, 7.0 g / L, 8.0 g / L, 9.0, or 10.0 g / L of a pH, O2, CO2, or dissolved CO2-sensitive resin, or a range defined by any two of the foregoing values. For example, in some embodiments, the culture container has or has about 0.01 g / L to 10.0 g / L, 0.01 g / L to 6.5 g / L, 0.01 g / L to 4.0 g / L, 0.01 g / L to 2.0 g / L, 0.01 g / L to 1.0 g / L, or 0.01 g / L to 0.10 g / L of a pH, O2, CO2, or dissolved CO2-sensitive resin. In some embodiments, the sensor is fluorescent. In some embodiments, the presence of Candida (such as Candida albicans or Candida glabrata), other yeasts, and / or other microorganisms in the biological sample causes an increase in the CO2 concentration in the gas in the headspace. In some embodiments, the presence of iron and / or copper in the microbial growth medium causes the CO2 concentration in the gas in the headspace to increase more rapidly than in a culture container containing a microbial growth medium without supplemented iron and / or copper. In some embodiments, the increased CO2 concentration in the gas in the headspace changes the pH of the microbial growth medium in the culture container.In some embodiments, a sensor monitors the CO2 concentration (e.g., provides a signal proportional thereto). In some embodiments, a sensor monitors the pH of a microbial growth medium contained within a culture vessel (e.g., provides a signal proportional thereto). In some embodiments, the pH sensor includes a pH-sensitive dye. In some embodiments, the pH sensor includes a plurality of pH-sensitive dyes. In some embodiments, the microbial growth medium and / or the pH sensor change to different colors to indicate a specific pH. In some embodiments, the pH sensor is a fluorescent probe. In some embodiments, the culture vessel is transparent. In some embodiments, the pH of a microbial growth medium bottle is monitored by a device. In some embodiments, the pH monitoring instrument includes a BACTEC FX. In some embodiments, detecting a change in pH and / or CO2 in the microbial growth medium indicates a systemic infection. In some embodiments, the biological sample is further processed to identify the microorganisms present in the biological sample. In some embodiments, an appropriate antibiotic regimen is administered to the subject for the identified microorganisms.

[0167] In some embodiments, the culture vessel includes a gas headspace. In some embodiments, the gas headspace contains CO2, O2, and N2. In some embodiments, the gas headspace contains only CO2, O2, and N2 (excluding contaminant gases). In some embodiments, the amount of O2 in the gas of the headspace is, is about, at least, at least about, not more than, or not more than about 25%, 30%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75% O2, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of O2 in the gas of the headspace is or is about 25% to 75%, 47% to 60%, or 52% to 56% O2. In some embodiments, the amount of CO2 in the gas of the headspace is, is about, at least, at least about, not more than, or not more than about 15%, 20%, 21%, 22%, 23%, 24%, 24.1%, 24.2%, 24.3%, 24.4%, 24.5%, 24.6%, 24.7%, 24.8%, 24.9%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 45% CO2, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of CO2 in the gas of the headspace is or is about 15% to 45%, 15% to 30%, 15% to 25%, 20% to 25%, or 24% to 25% CO2. In some embodiments, the amount of N2 in the gas of the headspace is the amount remaining after adding the amounts of O2 and CO2 disclosed herein. In some embodiments, the amount of N2 in the gas of the headspace is, is about, at least, at least about, not more than, or not more than about 0%, 5%, 10%, 15%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 28.1%, 28.3%, 28.3%, 28.4%, 28.5%, 28.6%, 28.7%, 28.8%, 28.9%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 40%, 45%, 50%, 55%, or 60% N2, or a range defined by any two of the foregoing values. For example, in some embodiments, the amount of N2 in the gas of the headspace is or is about 0% to 60%, 0% to 40%, 0% to 30%, 20% to 40%, or 20% to 30% N2. In some embodiments, the gas of the headspace contains, contains about, contains at least, contains at least about, contains not more than, or contains not more than about 25% to 75% O2, 15% CO2 to 45% CO2, and 0% to 60% N2, optionally wherein the gas of the headspace contains only CO2, O2, and N2 (excluding contaminant gases).In some embodiments, the gas in the headspace comprises, comprises about, comprises at least, comprises at least about, comprises no more than, or comprises no more than about 52% to 56% O2, 24% CO2 to 25% CO2, and 19% to 24% N2. In some embodiments, the gas in the headspace comprises, comprises about, comprises at least, comprises at least about, comprises no more than, or comprises no more than about 24% CO2, 47.5% O2, and 28.5% N2.

[0168] In some embodiments, adding iron and / or copper to a microbial growth medium as described herein results in an increased oxygen consumption by the microorganisms growing in the medium. In some embodiments, the oxygen concentration in the gas in the headspace is selected for a particular microorganism. In some embodiments, the microorganism is yeast. In some embodiments, the microorganism is selected from one or more of the following: Abiotrophia defectiva, Acinetobacter lwoffii, Actinobacillus actinomycetemcomitans, Aerococcus viridans, Alcaligenes faecalis, Bacillus subtilis, Candida albicans, Candida glabrata, Cardiobacterium hominis, Corynebacterium jeikeium, Cryptococcus neoformans, Eikenella corrodens, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Granulicatella adiacens, Haemophilus influenzae, Haemophilus influenzae, Haemophilus influenzae type a, Haemophilus influenzae type b, Haemophilus parainfluenzae, Kingella kingae, Klebsiella pneumoniae, Leuconostoc species, Micrococcus luteus, Neisseria gonorrhoeae, Neisseria meningitidis, Pediococcus acidilactici, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Rothia mucilaginosa, Saccharomyces cerevisiae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus sanguinis. In some embodiments, the culture vessel comprises a pH sensor. In some embodiments, the pH sensor is a dye. In some embodiments, the pH sensor is fluorescent. In some embodiments, the growth of microorganisms within a culture vessel containing a microbial growth medium alters the concentration of CO2 in the culture vessel. In some embodiments, the growth of microorganisms within a culture vessel containing a microbial growth medium increases the concentration of CO2 in the culture vessel. In some embodiments, increasing the concentration of CO2 within the microbial growth medium alters the pH of the enhanced microbial growth medium. In some embodiments, adding iron and / or copper to a microbial growth medium shortens the TTD of Candida (e.g., Candida albicans or Candida glabrata), other yeasts, and / or other microorganisms in a biological sample. In some embodiments, the biological sample is blood. In some embodiments, the biological sample is serum. In some embodiments, the TTD, percent recovery, DVE, false positive rate, antimicrobial growth support, or any combination thereof of a test microbial growth medium is tested.

[0169] In some embodiments, systems are disclosed for detecting the presence of microorganisms in a sample. In some embodiments, the system includes a microbial growth culture vessel as described herein. In some embodiments, the system for detecting the presence of microorganisms in a sample includes a detector for obtaining a signal from a sensor. In some embodiments, the system for detecting the presence of microorganisms in a sample includes a computer configured to determine whether the signal obtained by the detector indicates the presence of microorganisms in the microbial growth medium. In some embodiments, the system includes a BACTEC FX instrument.

[0170] In some embodiments, methods of culturing microbial organisms are disclosed. In some embodiments, the method includes inoculating the microbial growth medium disclosed herein with a biological sample and culturing the microorganisms in the sample in the microbial growth medium. In some embodiments, the biological sample is a blood, serum, plasma, or urine sample. In some embodiments, the biological sample is obtained from a septic subject. In some embodiments, the biological sample is obtained from a systemic inflammatory response syndrome (SIRS)-positive subject. In some embodiments, the inoculation step includes adding the sample to the microbial growth medium of a microbial growth culture vessel as described herein. In some embodiments, the microbial organism is Candida (e.g., Candida albicans or Candida glabrata), other yeast, and / or other microorganisms.

[0171] In some embodiments, methods for detecting the presence of microorganisms in a biological sample are disclosed. In some embodiments, the biological sample is a blood, serum, plasma, or urine sample. In some embodiments, the biological sample is obtained from a septic subject. In some embodiments, the biological sample is obtained from a subject positive for systemic inflammatory response syndrome (SIRS). In some embodiments, methods for detecting the presence of Candida (e.g., Candida albicans or Candida glabrata), other yeasts, and / or other microorganisms in a biological sample are disclosed. In some embodiments, the microorganisms are Atopobium parvulum, Acinetobacter lwoffii, Aggregatibacter actinomycetemcomitans, Aerococcus viridans, Alcaligenes faecalis, Bacillus subtilis, Candida albicans, Candida glabrata, Cardiobacterium hominis, Corynebacterium jeikeium, Cryptococcus neoformans, Eikenella corrodens, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Granulicatella adiacens, Haemophilus influenzae, Haemophilus influenzae, Haemophilus influenzae type a, Haemophilus influenzae type b, Haemophilus parainfluenzae, Kingella kingae, Klebsiella pneumoniae, Leuconostoc species, Micrococcus luteus, Neisseria gonorrhoeae, Neisseria meningitidis, Pediococcus acidilactici, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Rothia mucilaginosa, Saccharomyces cerevisiae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus sanguinis. In some embodiments, the volume of the biological sample is, is about, is at least, is at least about, is no more than, or is no more than about 0 mL, 0.001 mL, 0.01 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, 4 mL, 4.5 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, or 10 mL, or is a range defined by any two of the foregoing values. For example, in some embodiments, the volume of the biological sample is or is about 0 mL to 10 mL, 0 mL to 5 mL, 0 mL to 3 mL, 1 mL to 10 mL, 3 mL to 10 mL, or 3 mL to 5 mL. In some embodiments, the device having a shortened Candida TTD includes a vessel having a body portion. In some embodiments, the body portion of the vessel contains a microbial growth medium having a shortened Candida TTD and a gas in the headspace. In some embodiments, the microbial growth medium contains DI water, iron, copper, sucrose, dextrose, D-trehalose, yeast extract, L-glutamic acid, tryptic soy broth, sodium polyanetholesulfonate, menadione, pyridoxal hydrochloride, ferulic acid, sodium hydroxide, ascorbic acid, L-cysteine, and hemin.

[0172] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs when reading this disclosure. For the purposes of this disclosure, the following terms are explained below.

[0173] Unless the context indicates otherwise, the articles "a" and "an" are used herein to refer to one or more than one (e.g., at least one) grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0174] "About" means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length that varies by up to 10% relative to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

[0175] Throughout the specification, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" will be understood to imply the inclusion of the stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. "Consisting of" means including and limited to whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory and that no other elements are present. "Consisting essentially of" means including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present, depending on whether they materially affect the activity or action of the listed elements.

[0176] This disclosure uses affirmative language to describe various embodiments and generally discloses the subject matter of this disclosure. This disclosure also includes embodiments that exclude the subject matter in whole or in part, such as substances or materials, method steps and conditions, schemes or procedures.

[0177] In at least some of the previously described embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, unless such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described herein without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the disclosed subject matter.

[0178] Regarding the use of basically any plural and / or singular terms herein, those skilled in the art can appropriately translate the plural into the singular and / or the singular into the plural according to the context and / or application. For clarity, various singular / plural permutations may be set forth herein explicitly.

[0179] Those skilled in the art will understand that the terms commonly used herein, especially those used in the appended claims (e.g., the subject matter of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be construed as "including but not limited to", the term "having" should be construed as "having at least", the term "includes" should be construed as "including but not limited to", etc.). Those skilled in the art will further understand that if the intention is to refer to a specified number of claim recitations, such intention should be explicitly stated in the claims, and in the absence of such a statement, such intention will not exist. For example, for the sake of understanding, the appended claims may include the use of introductory phrases such as "at least one / at least a kind of" and "one or more / a kind or more kinds of" to introduce claim recitations. However, even when the same claim includes an introductory phrase such as "one or more / a kind or more kinds of" or "at least one / at least a kind of" and an indefinite article, such as "a / an" (e.g., "a / an" should be construed as meaning "at least one / at least a kind of" or "one or more / a kind or more kinds of"), the use of such phrases should not be construed as implying that the introduction of a claim recitation by the indefinite article "a / an" limits any particular claim containing such introduced claim recitation to an embodiment containing only one such recitation; the same is true for the use of a definite article for introducing a claim recitation. In addition, even if the specific number of the introduced claim recitation is explicitly stated, those skilled in the art will also recognize that such a statement should be construed as meaning at least the stated number (e.g., a mere statement of "two recitations" without any other modification means at least two recitations, or two or more recitations). In addition, in those cases where a conventional expression similar to "at least one of A, B, and C, etc." is used, generally such a construction is intended to have the conventional meaning that those skilled in the art will understand (e.g., "a system having at least one of A, B, and C" will include, but not be limited to, a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a conventional expression similar to "at least one of A, B, or C, etc." is used, generally such a construction is intended to have the conventional meaning that those skilled in the art will understand (e.g., "a system having at least one of A, B, or C" will include, but not be limited to, a system having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).Those skilled in the art will further understand that, whether in the specification, claims, or drawings, virtually any separated words and / or phrases that represent two or more alternative terms should be understood as contemplating the possibility of including one of such terms, any one of such terms, or both of such terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B". The use of "A and / or B" should be understood to include the possibilities of "A" or "B" or "A and B".

[0180] In addition, where features or aspects of the present disclosure are described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is also thereby described in terms of any single member or subgroup of members of the Markush group.

[0181] As will be understood by those skilled in the art, for any and all purposes, such as for providing a written description, all ranges disclosed herein also cover any and all possible subranges and combinations of such subranges. Any listed range can be readily considered to be sufficiently described and such that the same range can be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily broken down into lower third, middle third, and upper third, etc. As will also be understood by those skilled in the art, all language such as "at most", "at least", "greater than", "less than", etc. includes the recited numbers and refers to ranges that can then be broken down into subranges as discussed herein. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 - 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 - 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0182] Although various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting.

[0183] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are hereby incorporated by reference in their entirety and the subject matter thereof, and thereby form a part of this specification. To the extent that the publications and patents or patent applications incorporated by reference are inconsistent with the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such conflicting material.

[0184] Examples

[0185] Some aspects of the embodiments discussed herein are disclosed in more detail in the following examples, which are not intended to limit the scope of the disclosure in any way. Those skilled in the art will appreciate that many other embodiments also fall within the scope of the invention, as described herein and in the claims.

[0186] Example 1

[0187] Culture Medium Formulation and Bottling

[0188] In a representative embodiment, a culture medium is formulated to shorten the detection time to detection (TTD) of yeast and other microorganisms in BD Plus Aerobic / 26F BACTEC bottles. The culture medium comprises 20 L of DI water, 2 g of ammonium ferric citrate, 0.02 g of copper sulfate, 6.6 g of sucrose, 40 g of dextrose, 40 g of D-trehalose, 50 g of yeast extract, 10 g of L-glutamic acid, 550 g of TSB, 10 g of SPS, 0.01 g of menadione, 0.2 g of pyridoxal hydrochloride, 0.1 g of ferulic acid, 15.31 g of sodium hydroxide, 1 g of ascorbic acid, 0.01 g of L-cysteine, and 0.1 g of hemin. The culture medium further comprises 0.1875 g of sodium hydroxide and 20 mL of DI water for the hemin solution; 0.2 g of sodium hydroxide and 20 mL of water for the ferulic acid solution; and 0.78 mL of ethanol for the menadione solution. The hemin, menadione, and ferulic acid solutions are all prepared no more than 72 hours prior to preparing the culture medium and stored at about 2 - 8°C. The hemin solution is prepared by pipetting 20 mL of DI water into a 50 mL Falcon tube. Sodium hydroxide is added to the tube and vortexed. Once the sodium hydroxide is dissolved, hemin is added to the tube and vortexed. Once the hemin is dissolved, the solution is labeled with a 72-hour expiration date and stored in a refrigerator at about 2 - 8°C. The menadione solution is prepared by pipetting 780 μL of ethanol into a conical tube, adding a pre-weighed amount of menadione to the tube, covering the tube, and vortexing until dissolved. Once the menadione is dissolved, the tube is labeled with a 72-hour expiration date and stored in a refrigerator at 2 - 8°C. The ferulic acid solution is prepared by pipetting 20 mL of DI water into a 50 mL Falcon tube, adding sodium hydroxide, and vortexing until the sodium hydroxide is dissolved. Once dissolved, ferulic acid is added to the tube and the solution is vortexed until the ferulic acid is completely dissolved. Once dissolved, the tube is labeled with a 72-hour expiration date and stored in a refrigerator at about 2 - 8°C.

[0189] In this example, the bead bath was preheated to approximately 70 °C, the pH meter was turned on and calibrated. Bottles for containing the prepared culture medium were preheated in a water bath. 12 L of DI water, a magnetic stir bar, and a thermometer were added to a 45 L carboy container located on top of a magnetic heating plate. The mixture was stirred at approximately 200 - 300 rpm and brought to a temperature of 45 °C to 75 °C. The solution was maintained at 45 °C to 75 °C during the preparation period. Then sucrose, dextrose, D-trehalose, yeast extract, and L-glutamic acid were each added to the tank in sequence. 3 L of DI water was added to the container to rinse off any remaining added components on the container walls. Then TSB was slowly added to the 45 L container to minimize dust generation, and then an additional 3 L of DI water was added. The TSB was allowed to completely dissolve for over 10 minutes, and then SPS, menadione solution, pyridoxal hydrochloride, and ferulic acid solution were added to the container in sequence. The sides of the container were rinsed with 1 L of DI water, and then sodium hydroxide, ascorbic acid, L-cysteine, ferric ammonium citrate, and copper sulfate were added to the container. Then the temperature of the container was raised to 75 °C + / - 3 °C. The hemin solution was pipetted into the container. The container was rinsed with 1 L of DI water, and the solution was stirred until the hemin solution was completely dissolved.

[0190] In this example, 40 - 50 mL of the prepared culture medium is taken out from the container for pH testing and cooled to 55 °C. Once cooled, the pH of the culture medium is tested and the pH of the culture medium is adjusted using NaOH or HCl to increase or decrease the pH so that the pH of the cooled culture medium is 7.70 to 7.80. After bottling, autoclaving and storing for about 1 to 7 days, after adding a Plus Aerobic gas mixture to the headspace, the above pH is converted to a room temperature pH of 7.1 - 7.4. The Plus Aerobic gas mixture contains 24% CO2, 47.5% O2 and 28.5% N2. To bottle the culture medium, start the culture medium dispenser and allow it to run until the culture medium is continuously dispensed at 67 °C to 78 °C. The preheated culture medium filled bottles are taken out one by one from the bead bath, the bottles are strategically added to and taken out from the bead bath to ensure that each bottle has a similar preheating time. The prepared culture medium and a Plus Aerobic gas mixture of about 5 - 10 psi are dispensed into the preheated filled bottles using a 3.5” F nozzle and a hold time of 5 seconds. A rubber stopper is placed in the bottle to indicate how far the nozzle can enter the bottle without disturbing the resin. The nozzle tip should reach just far enough to pass through the bottleneck. It is crucial to have a space between the bottle mouth and the nozzle so that the gas can be blown out. Each bottle is filled with 1.75 g of a CO2 sensor, 4.6 g of resin and 30 mL of the culture medium. Then the filled bottles are capped and sealed with an automated crimper. Then the filled bottles are sterilized using an air over pressure autoclave. The filled sterile bottles are stored at room temperature.

[0191] Time to Detection (TTD) Test

[0192] The prepared culture medium is tested using Plus Aerobic / 26F BACTEC bottles manufactured within one week of the culture medium preparation as a control. Biological tests include time to detection (TTD), percentage recovery (sensitivity), false positive rate, false negative rate, delayed vial entry (DVE), and antimicrobial growth support. TTD tests are performed using 43 organisms with a target CFU of 10 - 100 in blood volumes of 0 mL or 0.5 mL (depending on the organism), 3 mL, and 10 mL. The tests are repeated three times. One day before the test, the bottles are placed on the shelf and labeled, and the organisms are subcultured accordingly. Figure 1A list of the test organisms, strains, media, and growth conditions is provided. Each organism to be tested is grown overnight on the appropriate plate medium. The purity of each organism is carefully examined. If there are contaminants or the colonies have an unusual appearance, Gram staining and / or identification are performed to confirm the identity. The organism is re-cultured. 0.1 mL of the final organism dilution is dispensed and plated on the appropriate agar for plate counts. Using aseptic technique, with the appropriate syringe and needle, 0.5 mL, 3 mL, or 10 mL of bagged human blood is added to each bottle containing 30 mL of medium. Paired groups are inoculated with blood from the same donor unit simultaneously. Then, 0.1 mL of the appropriate organism is inoculated into the bottles. After inoculation, the bottles are loaded onto the BACTEC FX instrument using a standard protocol length of 120 hours. The paired bottles enter adjacent positions in the FX simultaneously. There are no relevant differences in the technical specifications of the TTD test compared to the Plus Aerobic / 26F BACTEC medium determined by Wilcoxon analysis. The TTD is evaluated by Wilcoxon paired analysis and comparison of the interval-based median. Only paired bottles in which growth is detected in both bottles within the 120-hour protocol are included. The median TTD must have no statistically detectable difference, i.e., a p-value less than 0.05, or under the test conditions where a statistically relevant detectable difference occurs, they should not favor the Plus Aerobic / 26F BACTEC medium. A relevant difference is defined as a time difference in detection of no more than 10% when favoring the Plus Aerobic / 26F BACTEC medium. The pass criteria are based on the collective performance of all organisms, but individual organisms that fall outside the 10% difference limit are discarded. The results of this experiment are listed in Figure 2 . The results of each biological test have results acceptable according to the criteria listed in each technical standard. There are several organisms that do not meet the 10% difference criteria. The individual organisms that did not pass the acceptance criteria are: Acinetobacter lwoffii, Haemophilus parainfluenzae, Neisseria meningitidis, Rothia mucilaginosa, Stenotrophomonas maltophilia, and Streptococcus pneumoniae. In some cases, the organisms are re-tested with additional strains. In 3 mL of blood, Acinetobacter lwoffii had a significantly longer TTD, favoring the control. The other two blood volumes had an average % difference in TTD that favored the new medium formulation. The results of this experiment are listed in Figure 3 . Due to suspected inoculation error, the organisms were repeated. In the repeat (results in light gray), the results for 3 mL favored the newly formulated medium bottles. The Haemophilus parainfluenzae results showed a significantly longer TTD in the bottles containing 10 mL of blood. The results of this experiment are shown in Figure 4In. The organism was repeated in all 3 blood volumes. The repeated results (light gray) for 10 mL of blood still favored the existing medium. It was expected that Haemophilus would exhibit a longer TTD in bottles containing the newly formulated medium due to the increased O2 and its sensitivity to high levels of oxygen. Three additional Haemophilus parainfluenzae strains were tested. The results of these experiments are listed in Figure 5 In. One strain showed a preference for PlusAerobic / 26F BACTEC medium in all 3 blood volumes. A second strain showed equivalent performance between Plus Aerobic / 26F BACTEC medium and the new medium formulation, and the third strain's results were split between the two medium types. Additionally, as the oxygen level decreased, improved performance was expected in bottles with the formulated medium. For 0.5 mL and 3 mL of blood, Neisseria meningitidis had a >10% difference in TTD, favoring Plus Aerobic / 26F BACTEC medium. The organism was repeated. The repeated results for 0.5 mL still did not meet the acceptance criteria by standard, but the repeated results for 3 mL were acceptable. The results of this experiment are listed in Figure 6 In. The TTD of this organism was also expected to improve as the oxygen level in the headspace gas decreased. The initial results for Rothia mucilaginosa showed a significantly longer TTD in bottles without blood and in bottles with 3 mL of blood. The organism was repeated. After retesting, bottles containing the new medium formulation had a significantly longer TTD for all blood volumes. The results of these experiments are shown in Figure 7 In. Three additional Rothia mucilaginosa strains were tested. The results of this experiment are listed in Figure 8 In. Results highlighted in gray did not pass the 10% acceptance criteria. Some results appeared to be outliers due to contamination or inoculation error. The new medium formulation showed better recovery in one strain. The average for all strains was within 10% of Plus Aerobic / 26F BACTEC. In the initial test, Stenotrophomonas maltophilia had a significantly longer TTD in all blood volumes. The results of these experiments are shown in Figure 9 In. The repeated results showed a significantly longer TTD in bottles without blood and in bottles with 3 mL of blood. The results were concerning because Stenotrophomonas maltophilia harbors known resistance to multiple antimicrobial therapies. Five additional strains were selected for testing. The results of these experiments are listed in Figure 10Among them. The average TTD for each strain and blood volume tested was below the 10% standard. The ATCC strains used for testing reacted differently to the newly prepared medium than the other strains tested and may not be representative of clinical strains and their TTD in the new medium formulation. The Streptococcus pneumoniae results showed a significantly longer TTD at 0 mL. This organism was repeated using all 3 blood volumes. The results of these experiments are listed in Figure 11 Among them. When repeated, the results were acceptable except for one repeat at 0 mL. This result was most likely due to an inoculation error. Three additional Streptococcus pneumoniae strains were tested as part of the organism list. The results of these experiments are listed in Figure 12 Among them. Data from this experiment indicated that the new medium formulation had equivalent enhanced performance when comparing TTD to the Plus Aerobic / 26F BACTEC medium containing no more than approximately 0.2 ppm iron and no more than approximately 0.025 ppm copper.

[0193] Percentage Recovery

[0194] Recovery tests were performed using 15 organisms with target CFUs of 0 - 1 and 1 - 10 in blood volumes of 0 mL or 0.5 mL (depending on the organism), 3 mL, and 10 mL. The tests were repeated 3 times. The organisms tested in these experiments are listed in Figure 13 Among them. One day prior to testing, the bottles were placed on the shelf and labeled, and the organisms were subcultured accordingly. Each organism to be tested was grown overnight on the appropriate plate medium. The purity of each organism was carefully examined. If contaminants were present or the colonies had an unusual appearance, Gram staining and / or identification was performed to confirm the identity. 0.1 mL of the final organism dilution was dispensed and plated on the appropriate agar for plate counts. Using aseptic technique, 0.5 mL, 3 mL, or 10 mL of bagged human blood was added to each bottle containing 30 mL of medium using an appropriate syringe and needle. A paired group was inoculated with blood from the same donor unit simultaneously. The bottles were inoculated with 0.1 mL of the appropriate organism. After inoculation, the bottles were loaded onto the BACTEC FX instrument using a standard protocol length of 120 hours. The paired bottles were placed in adjacent positions in the FX simultaneously. The technical criteria for percent recovery had no relevant differences from the existing medium. Recovery was evaluated by McNemar's chi - square test at the 95% confidence level with no statistical difference (P < 0.5). Any statistical difference in favor of the control was to be determined as relevant, which required determination of clinical relevance. The results of the McNemar's chi - square analysis are listed in Figure 14 Among them. Each p - value > 0.05, indicating no significant difference in the results. The results of % recovery were acceptable.

[0195] False Positive Rate

[0196] The false positive rate determination was carried out by inoculating the medium with 2 mL, 4 mL, 6 mL, 8 mL or 10 mL of freshly drawn sterile blood. Each blood volume was tested 8 times using freshly prepared medium and Plus Aerobic / 26F BACTEC bottles containing 30 ml of microbial growth medium. Each paired set received blood from the same donor simultaneously. After adding the blood, the bottles were loaded onto the BACTEC FX instrument using a standard protocol length of 120 hours. The paired bottles entered adjacent positions on the FX simultaneously. This technical specification is equivalent to the false positive rate of the Plus Aerobic / 26F BACTEC medium. The possibility of an increased false positive result due to differences in signal output was evaluated by producing expected negative bottles with different levels of fresh blood and demonstrating no difference in the observed rate of false positive determination (chi-square analysis). The results of the chi-square analysis are listed in Figure 15 None of the bottles inoculated with fresh blood had positive results, so the p-value could not be calculated.

[0197] Delayed Vial Entry (DVE)

[0198] The three DVE conditions tested were as follows: 1) the inoculated bottles were incubated at 35 ± 1 °C for 12 hours; 2) the inoculated bottles were incubated on the laboratory bench at room temperature (25 ± 2.5 °C) for 24 hours; and 3) the inoculated bottles were incubated on the laboratory bench at room temperature (25 ± 2.5 °C) for 36 hours. Each DVE condition was tested with 14 organisms with a target CFU of 10 - 100 in blood volumes of 3 mL and 10 mL. The tests were repeated 3 times. At Figure 16The organisms used in the DVE test are listed. One day before the test, the bottles are placed on the shelf and labeled, and the organisms are subcultured accordingly. Each organism to be tested is grown overnight on the appropriate plate medium. The purity of each organism is carefully examined. If there are contaminants or the colonies have an unusual appearance, Gram staining and / or identification is performed to confirm the identity. 0.1 mL of the final organism dilution is dispensed and plated on the appropriate agar for plate counting. Using aseptic technique, 3 mL or 10 mL of bagged human blood is added to each bottle containing 30 mL of microbial growth medium using the appropriate syringe and needle. Paired groups are inoculated with blood from the same donor unit. The bottles are inoculated with 0.1 mL of the appropriate organism. After inoculation, the bottles are loaded onto the BACTEC FX instrument using the standard protocol length of 120 hours. The paired bottles enter adjacent positions on the FX simultaneously. The technical specifications are equivalent to the Plus Aerobic / 26F BACTEC medium or there are no relevant differences from the Plus Aerobic / 26F BACTEC. The DVE recovery is evaluated by McNemar's chi-square test at a 95% confidence level with no difference (p ≤ 0.05). The correlation is defined as a statistically lower DVE recovery in the newly formulated medium compared to the reference medium using a p-value < 0.05. The results of the McNemar's chi-square analysis are listed in Figure 17 . The p-value at 36 hours at room temperature was 1.000, indicating no significant difference. The p-values at 12 hours at 35 °C and 24 hours at room temperature could not be calculated because each bottle had a positive result. The DVE results passed the validation and confirmation criteria.

[0199] Antimicrobial Growth Support

[0200] Two conditions are tested with 9 organisms. The target CFU is 10 - 100. 14 antibiotics are used. A blood volume of 10 mL is used. The test is repeated three times. Figure 18Lists the organisms and antibiotics used with the test levels. One day before testing, place the bottles on the shelf and label them, and subculture the organisms accordingly. Prepare the original stock solution and working stock solution of the antibiotics and store them appropriately. Each organism to be tested is grown overnight on the appropriate plate medium. Carefully examine the purity of each organism. If there are contaminants or the colonies have an unusual appearance, perform a Gram stain and / or identification to confirm the identity. Dispense 0.1 mL of the final organism dilution and spread it on the appropriate agar for plate counting. Using aseptic technique, add 3 mL or 10 mL of bagged human blood to each bottle containing 30 mL of microbial growth medium using an appropriate syringe and needle. Inoculate paired groups with blood from the same donor unit at the same time. Inoculate the bottles with 0.1 mL of the appropriate organism and 0.5 mL of the appropriate antibiotic working solution. After inoculation, load the bottles onto the BACTEC FX instrument using the standard protocol length of 120 hours. The paired bottles enter adjacent positions in the FX at the same time. The technical specification is equivalent antimicrobial removal in the newly formulated medium compared to the Plus Aerobic / 26F BACTEC. No relevant difference from the current medium is defined by one of the following test result conditions: 1) The number of microorganisms detected in the newly formulated medium is greater than the number detected in the Plus Aerobic / 26F BACTEC medium; or 2) If test condition 1 is not met, the McNemar chi-square test results in a p-value > 0.05. The results of the McNemar chi-square analysis are listed in Figure 19 . Condition 1 was not met. One of the 6 bottles was positive for Escherichia coli and imipenem. This bottle was the Plus Aerobic / 26F BACTEC control. Two of the 6 bottles were positive for Streptococcus pneumoniae and vancomycin. These two bottles were the Plus Aerobic / 26F BACTEC controls. Our resin has poor absorption of vancomycin and imipenem, and this is a known test risk. When condition 1 is not met, calculate the p-value. The p-value was 0.250, greater than 0.05, indicating no significant difference. The antimicrobial growth support test passed the validation and confirmation criteria. The data from this example clearly show that the new medium formulation has enhanced performance when comparing the TTD with the Plus Aerobic / 26F BACTEC. In addition, compared to the Plus Aerobic / 26F BACTEC, the new medium formulation has equivalent enhanced false positive rates, percentage recoveries, DVE, and antimicrobial growth support.

[0201] Example 2

[0202] In a representative example of the present disclosure, the ratio of iron to oxygen is optimized to promote improved TTD of yeast in the medium while maintaining a shelf life equivalent to Plus Aerobic / 26F BACTEC. TTD testing was performed using 28 organisms with a target CFU of 10 - 100 in blood volumes of 0 mL or 0.5 mL (depending on the organism), 3 mL, and 10 mL. The organisms tested are shown in Figure 20 . Four different concentrations of iron provided as ammonium ferric citrate (0 g / L, 0.01 g / L, 0.035 g / L, and 0.06 g / L) in the medium as described in Example 1 were tested, as well as three different concentrations of oxygen (47%, 54%, and 60%). Figure 21 The combinations of the conditions tested are shown. The tests were repeated three times. One day prior to testing, the bottles were placed on the shelf and labeled, and the organisms were subcultured accordingly. Each organism to be tested was grown overnight on an appropriate plate medium. The purity of each organism was carefully examined. If there were contaminants or the colonies had an unusual appearance, Gram staining and / or identification was performed to confirm the identity. The organisms were then recultured. 0.1 mL of the final organism dilution was aliquoted and spread on an appropriate agar for plate counting. Using aseptic technique, 0 mL, 0.5 mL, 3 mL, or 10 mL of bagged human blood was added to each bottle containing 30 mL of microbial growth medium using an appropriate syringe and needle. A paired group was inoculated with blood from the same donor unit simultaneously. The bottles were then inoculated with 0.1 mL of the appropriate organism. After inoculation, the bottles were loaded onto a BACTEC FX instrument using a standard protocol length of 120 hours. The paired bottles entered adjacent positions in the FX simultaneously. Figure 22 A histogram showing the TTD of microorganisms in blood volumes of 0 mL, 0.5 mL, 3 mL, and 10 mL is shown. Although there was some variation in TTD with different blood volumes, the average TTD for all blood volumes and organisms was approximately 24 - 27 hours. Figure 23 A histogram showing the TTD of five different classes of microorganisms (GC / Haem or Neisseria / Haemophilus, GNB or Gram - negative bacilli, GPB / GNCB or Gram - positive bacilli / Gram - negative coccobacilli, GPC or Gram - positive cocci, and yeast) at three concentrations of O2 (47%, 54%, and 60%) in the gas headspace is shown. The vertical dashed line at 30 - hour TTD in each panel represents the approximate TTD for all organisms. Figure 24Shows a histogram of the TTD of GC / Haem, GNB, GPB / GNCB, GPC, and yeast in freshly prepared media containing each of four different concentrations of iron (0 g / L, 0.01 g / L, 0.035 g / L, and 0.06 g / L). The vertical dashed lines at 30 h TTD in each panel represent the approximate TTD for all organisms. Figure 25 Shows a dot plot of the TTD of GC / Haem, GNB, GPB / GNCB, GPC, and yeast microorganisms in microbial growth media with blood volumes of 0 mL / 0.5 mL, 3 mL, and 10 mL and containing 0 g / L, 0.01 g / L, 0.035 g / L, and 0.06 g / L iron. The vertical dashed lines at 30 h TTD in each panel represent the approximate TTD for all organisms. Increasing the iron concentration in the media from 0 to 0.06 g / L shortened the TTD of yeast and other microorganisms, with an optimal iron concentration of approximately 0.035 g / L (light gray dots). Figure 26 Shows a dot plot of the TTD of GC / Haem, GNB, GPB / GNCB, GPC, and yeast microorganisms in the headspace of microbial growth media bottles with blood volumes of 0 mL / 0.5 mL, 3 mL, and 10 mL and containing 47%, 54%, or 60% O2. The vertical dashed lines at 30 h TTD in each panel represent the approximate TTD for all organisms. Increasing the oxygen concentration in the gas in the headspace of the microbial growth media bottles from 47% to 60% O2 shortened the TTD of yeast and other microorganisms, with an optimal headspace O2 concentration of approximately 54% (light gray dots). Figure 27 Shows a dot plot of the TTD of each of 28 organisms tested in microbial growth media with blood volumes of 0 mL / 0.5 mL, 3 mL, and 10 mL and containing 0 g / L, 0.01 g / L, 0.035 g / L, and 0.06 g / L iron. The vertical dashed lines at 30 h TTD in each panel represent the approximate TTD for all organisms. Increasing the iron concentration in the microbial growth media from 0 to 0.06 g / L shortened the TTD of Candida glabrata, Candida albicans, and other microorganisms, with an optimal iron concentration of approximately 0.035 g / L (light gray dots). Figure 28A dot plot is shown depicting the TTD of each of 28 organisms tested in the gas in the headspace of microbial growth medium bottles with blood volumes of 0 mL / 0.5 mL, 3 mL, and 10 mL and containing 47%, 54%, or 60% O2. The vertical dashed lines at 30 hours TTD in each panel represent the approximate TTD of all organisms. Increasing the oxygen concentration in the gas in the headspace of the microbial growth medium bottles from 47% to 60% O2 shortened the TTD of Candida glabrata, Candida albicans, and other microorganisms, with an optimal gas headspace O2 concentration of approximately 54% (light gray dots). Figure 29 A summary histogram is shown of the TTD of yeast in microbial growth medium containing 0 g / L, 0.01 g / L, 0.035 g / L, and 0.06 g / L iron. Figure 30 A summary histogram is shown of the TTD of all other microorganisms tested in microbial growth medium containing 0 g / L, 0.01 g / L, 0.035 g / L, and 0.06 g / L iron. Figure 31 A histogram is shown depicting the improved TTD of yeast 2901 compared to the TTD of all other microorganisms 2902 in microbial growth medium containing 0 g / L, 0.01 g / L, 0.035 g / L, and 0.06 g / L iron. Increasing the iron concentration in the medium from 0 to 0.06 g / L shortened the TTD of yeast and other microorganisms, with an optimal iron concentration of approximately 0.035 g / L. Figures 33 - 55 A bivariate fit and summary histogram are shown of the TTD of microorganisms in Plus Aerobic / 26F BACTEC; freshly prepared medium in microbial growth medium bottles with 47%, 54%, and 60% O2 in the gas headspace; and freshly prepared microbial growth medium containing 0 g / L, 0.01 g / L, 0.035 g / L, and 0.06 g / L iron in microbial growth medium bottles with 47%, 54%, and 60% O2 in the gas headspace. Data from each experiment were organized for statistical testing by general linear model analysis of variance (ANOVA). Blood volumes of 0 mL or 0.5 mL, 3 mL, and 10 mL were recorded as 0 mL, 3 mL, and 10 mL to help balance the design. Additional models were evaluated to keep the blood volume constant. The effects and parameters of the ANOVA are shown in Figure 56 and Figure 57 . Figure 56 Results are shown demonstrating the effect of the ANOVA analysis of test conditions on TTD. Figure 57The ANOVA parameter estimates and the results of the prediction equation are shown. Only the yeast species is expected to show a significant effect of added iron. Other categories are monitored to ensure no adverse effects. The results indicate that iron reduces the TTD of yeast and may also be beneficial to other organisms.

Claims

1. A microbial growth medium that provides an increased growth rate of Candida, said microbial growth medium comprising: iron at a concentration of from about 1 μM to about 400 μM; and optionally copper at a concentration not exceeding about 700 μM; wherein the growth rate of Candida cultured in said microbial growth medium is increased as compared to the same microbial growth medium without said iron and copper.

2. The microbial growth medium according to claim 1, wherein the detection time to detection (TTD) of Candida cultured in said microbial growth medium is shortened as compared to the same microbial growth medium without said iron and copper.

3. The microbial growth medium according to any one of the preceding claims, wherein the shortened TTD of Candida cultured in said microbial growth medium is determined by: adding 0.5 mL, 3 mL, and 10 mL of blood to culture vessels containing the microbial growth medium with and without said iron and copper; inoculating said culture vessels with 0.1 mL of Candida culture to provide 10 - 100 CFU of Candida per vessel; culturing said Candida at about 30°C to 37°C for up to 120 hours; monitoring the signal indicating the presence of Candida in said culture vessels; and determining the TTD of said Candida in the microbial growth medium with and without said iron and copper.

4. The microbial growth medium according to any one of the preceding claims, wherein the Candida is Candida albicans ( Candida albicans ) or Candida glabrata ( Candida glabrata ).

5. The microbial growth medium according to any one of the preceding claims, wherein said microbial growth medium comprises or consists of about 0 g / L, 0.01 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, or 1.0 g / L of yeast nitrogen base, or a yeast nitrogen base within a range defined by any two of the foregoing values.

6. The microbial growth medium according to any one of the preceding claims, wherein said microbial growth medium comprises less than 0.01% w / v of yeast nitrogen base.

7. The microbial growth medium according to any one of the preceding claims, wherein said yeast nitrogen base does not contain histidine, methionine, and tryptophan.

8. The microbial growth medium according to any one of the preceding claims, wherein the microbial growth medium comprises or consists of about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 28 μM, 50 μM, 75 μM, 100 μM, 125 μM, 150 μM, 170 μM, 175 μM, 200 μM, 225 μM, 250 μM, 275 μM, 300 μM, 325 μM, 350 μM or 400 μM of iron, or a range defined by any two of the foregoing values, optionally 1 μM to 400 μM, 1 μM to 350 μM, 1 μM to 200 μM, 1 μM to 100 μM, 1 μM to 50 μM, 2 μM to 400 μM, 2 μM to 350 μM, 25 μM to 400 μM, 25 μM to 325 μM, 25 μM to 200 μM, 25 μM to 100 μM or 25 μM to 75 μM of iron.

9. The microbial growth medium according to any one of the preceding claims, wherein the concentration of iron is about 28 μM.

10. The microbial growth medium according to any one of the preceding claims, wherein the concentration of iron is about 100 μM.

11. The microbial growth medium according to any one of the preceding claims, wherein the concentration of iron is about 170 μM.

12. The microbial growth medium according to any one of the preceding claims, wherein the iron is ferric iron.

13. The microbial growth medium according to any one of the preceding claims, wherein the iron is provided as ammonium ferric citrate, ferric chloride, ferric sulfate, or ferric nitrate.

14. The microbial growth medium according to any one of the preceding claims, wherein the microbial growth medium comprises or consists of about 0 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 75 μM, 100 μM, 150 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 650 μM or 700 μM of copper, or a range defined by any two of the foregoing values, optionally 0 μM to 700 μM, 0 μM to 650 μM, 0 μM to 500 μM, 0 μM to 300 μM, 0 μM to 100 μM, 0 μM to 50 μM, 0 μM to 25 μM, 0.1 μM to 100 μM, 0.1 μM to 50 μM, 0.1 μM to 25 μM, 0.5 μM to 650 μM, 0.5 μM to 300 μM, 0.5 μM to 100 μM, 0.5 μM to 50 μM or 0.5 μM to 25 μM of copper.

15. The microbial growth medium according to any one of the preceding claims, wherein the concentration of copper is about 3 μM.

16. The microbial growth medium according to any one of the preceding claims, wherein the copper is divalent copper.

17. The microbial growth medium according to any one of the preceding claims, wherein the copper is provided as copper sulfate, copper chloride, copper nitrate, copper bromide, copper chlorate and / or copper(II) gluconate.

18. The microbial growth medium according to any one of the preceding claims, wherein the microbial growth medium is an aqueous liquid growth medium, and the aqueous liquid growth medium further comprises sucrose, dextrose, D - trehalose, yeast extract, L - glutamic acid, tryptic soy broth (TSB), sodium polyanetholesulfonate (SPS), menaquinone, pyridoxal hydrochloride, ferulic acid, sodium hydroxide, ascorbic acid, L - cysteine, hemin.

19. The microbial growth medium according to any one of the preceding claims, wherein the microbial growth medium comprises, in water or in an aqueous solution, about 1.0 g / L to 10.0 g / L yeast extract, 0.15 g / L to 1.5 g / L sucrose, 1.0 g / L to 10.0 g / L dextrose, 1.0 g / L to 10.0 g / L D-trehalose, 0.001 g / L to 1.5 g / L L-glutamic acid, 0.00001 g / L to 0.01 g / L L-cysteine, 0.5 g / L to 15 g / L TSB, 0.001 g / L to 1.5 g / L SPS, 0.00001 g / L to 0.01 g / L menaquinone, 0.0001 g / L to 1 g / L pyridoxal hydrochloride, 0.0001 g / L to 1 g / L ferulic acid, 0.1 g / L to 10 g / L sodium hydroxide, 0.001 g / L to 1.0 g / L ascorbic acid, and 0.0001 g / L to 1 g / L hemin chloride.

20. The microbial growth medium according to any one of the preceding claims, wherein the microbial growth medium comprises, in water or in an aqueous solution, about 0.001 g / L to 0.15 g / L iron, 0.0001 g / L to 0.001 g / L copper, 3.0 g / L to 5.0 g / L yeast extract, 0.15 g / L to 1.5 g / L sucrose, 2.5 g / L to 3.5 g / L dextrose, 1.5 g / L to 3.5 g / L D-trehalose, 0.5 g / L to 1 g / L L-glutamic acid, 0.0001 g / L to 0.0012 g / L L-cysteine, 0.5 g / L to 60 g / L TSB, 0.5 g / L to 1.5 g / L SPS, 0.0004 g / L to 0.0012 g / L menaquinone, 0.005 g / L to 0.025 g / L pyridoxal hydrochloride, 0.001 g / L to 0.1 g / L ferulic acid, 0.5 g / L to 3 g / L sodium hydroxide, 0.01 g / L to 0.25 g / L ascorbic acid, and 0.001 g / L to 0.1 g / L hemin chloride.

21. The microbial growth medium according to any one of the preceding claims, wherein the microbial growth medium comprises or consists essentially of about 0.01 g / L iron, 0.001 g / L copper, 0.528 g / L sucrose, 3.2 g / L dextrose, 3.2 g / L D - trehalose, 4 g / L yeast extract, 0.8 g / L L - glutamic acid, 44 g / L TSB, 0.8 g / L SPS, 0.0008 g / L menaquinone, 0.016 g / L pyridoxal hydrochloride, 0.008 g / L ferulic acid, 1.225 g / L sodium hydroxide, 0.08 g / L ascorbic acid, 0.0008 g / L L - cysteine, and 0.008 g / L hemin chloride.

22. The microbial growth medium according to any one of the preceding claims, wherein the microbial growth medium comprises or consists essentially of about 0.01 g / L iron, 0.001 g / L copper, 0.33 g / L sucrose, 2 g / L dextrose, 2 g / L D - trehalose, 2.5 g / L yeast extract, 0.5 g / L L - glutamic acid, 27.5 g / L TSB, 0.5 g / L SPS, 0.00005 g / L menaquinone, 0.01 g / L pyridoxal hydrochloride, 0.005 g / L ferulic acid, 0.7655 g / L sodium hydroxide, 0.05 g / L ascorbic acid, 0.0005 g / L L - cysteine, and 0.005 g / L hemin chloride.

23. The microbial growth medium according to any one of the preceding claims, wherein the microbial growth medium is capable of supporting the growth of one or more microbial species selected from the following: Atopobium defectivum( Abiotrophia defectiva ), Acinetobacter lwoffii( Acinetobacter lwoffii ), Aggregatibacter actinomycetemcomitans( Agreggatibacte ractinomycetemcomitans ), Aerococcus viridans( Aerococcus viridins ), Alcaligenes faecalis, Bacillus subtilis( Bacillus subtilis ), Candida albicans, Candida glabrata, Cardiobacterium hominis( Cardiobacterium hominis ), Corynebacterium jeikeium( Corynebacterium jeikeium ), Cryptococcus neoformans( Cryptococcus neoformans ), Eikenella corrodens( Eikinella corrodens ), Enterobacter cloacae( Enterobacter cloacae ), Enterococcus faecalis( Enterococcus faecalis ), Escherichia coli( Escherichia coli ), Granulicatella adiacens( Granulicatella adiacens ), Haemophilus influenzae( Haemophilus influenzae ), Haemophilus influenzae, Haemophilus influenzae type a( Haemophilus influenzae type a ), Haemophilus influenzae type b( Haemophilus influenzae type b ), Haemophilus parainfluenzae( Haemophilus parainfluenzae ), Kingella kingae, Klebsiella pneumoniae( Klebsiella pneumoniae ), Leuconostoc species( Leuconostoc species ), Micrococcus luteus( Micrococcus luteus ), Neisseria gonorrhoeae( Neisseria gonorrhoeae ), Neisseria meningitidis( Neisseria meningitidis ), Pediococcus acidilactici( Pediococcus acidilactici ), Proteus mirabilis( Proteus mirabilis ), Providencia stuartii( Providencia stuartii ), Pseudomonas aeruginosa( Pseudomonas aeruginosa ), Rothia mucilaginosa( Rothia mucilaginosa ), Saccharomyces cerevisiae( Saccharomyces cerevisiae ), Staphylococcus aureus( Staphylococcus aureus ), Staphylococcus epidermidis( Staphylococcus epidermidis ), Stenotrophomonas maltophilia( Stenotrophomonas maltophilia ), Streptococcus agalactiae( Streptococcus agalactiae ), Streptococcus pneumoniae( Streptococcus pneumoniae ) Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes Streptococcus pyogenes ) and Streptococcus sanguinis Streptococcus sanguinis ).

24. The microbial growth medium according to any one of the preceding claims, wherein the growth rate of Candida is increased by, is about, is at least, is at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75% or 80%, or is a range defined by any two of the foregoing values.

25. The microbial growth medium according to any one of the preceding claims, wherein the TTD of Candida is shortened by, is about, is at least, is at least about, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 75% or 80%, or is a range defined by any two of the foregoing values.

26. The microbial growth medium according to any one of the preceding claims, wherein the TTD of Candida is shortened by, is about, is at least, is at least about, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours or 72 hours, or is a range defined by any two of the foregoing values.

27. A culture container for detecting microbial growth, the container comprising: The microbial growth medium according to any one of the preceding claims; and A sensor for monitoring a parameter of the microbial growth medium, the parameter indicating microbial growth in the microbial growth medium.

28. The culture container according to claim 27, wherein the sensor is separated from the contents of the microbial growth medium by a permeable membrane.

29. The culture container according to claim 27 or 28, wherein the monitored parameter is pH, O2 and / or CO2.

30. The culture container according to any one of claims 27 to 29, wherein the sensor comprises a pH sensor.

31. The culture container according to claim 30, wherein the pH sensor comprises a fluorescent, phosphorescent or colorimetric pH-responsive reagent.

32. The culture vessel according to any one of claims 27 to 31, wherein the sensor comprises an O2 sensor.

33. The culture vessel according to claim 32, wherein the pH sensor comprises a fluorescent, phosphorescent or colorimetric O2-responsive reagent.

34. The culture vessel according to any one of claims 27 to 33, wherein the sensor comprises a CO2 sensor.

35. The culture vessel according to claim 34, wherein the CO2 sensor comprises a fluorescent, phosphorescent or colorimetric pH-responsive reagent.

36. The culture vessel according to any one of claims 27 to 35, wherein the sensor comprises a pH, O2 and / or CO2-sensitive resin.

37. The culture vessel according to claim 36, wherein the culture vessel contains, is about, is at least, is at least about, does not exceed or does not exceed about, 0.01 g / L, 0.025 g / L, 0.05 g / L, 0.075 g / L, 0.1 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L, 3.0 g / L, 4.0 g / L, 5.0 g / L, 6.0 g / L, 7.0 g / L, 8.0 g / L, 9.0 g / L or 10.0 g / L, or a pH and / or CO2-sensitive resin in a range defined by any two of the foregoing values.

38. The culture vessel according to any one of claims 27 to 37, wherein the container further comprises a headspace volume containing a gas mixture of O2, CO2 and N2.

39. The culture vessel according to claim 38, wherein the gas in the headspace comprises: from about 25% to about 75% O2, from about 15% to about 45% CO2, and from about 0% to about 40% N2.

40. The culture vessel according to claim 38 or 39, wherein the gas in the headspace contains, contains about, contains at least, contains at least about, contains less than or contains less than about, 25%, 30%, 40%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, or O2 in a range defined by any two of the foregoing values.

41. The culture vessel according to claim 38 or 39, wherein the gas in the headspace contains from about 47% to 60% O2.

42. The culture vessel according to claim 38 or 39, wherein the gas in the headspace contains from about 52% to 56% O2.

43. The culture container according to any one of claims 38 to 42, wherein the gas in the headspace comprises, comprises about, comprises at least, comprises at least about, comprises less than or comprises less than about, 15%, 20%, 21%, 22%, 23%, 24%, 24.1%, 24.2%, 24.3%, 24.4%, 24.5%, 24.6%, 24.7%, 24.8%, 24.9%, 25%, 26%, 27%, 28%, 29%, 30%, 40%, 45%, or CO2 in a range defined by any two of the foregoing values.

44. The culture container according to any one of claims 38 to 42, wherein the gas in the headspace comprises about 20% to about 30% CO2.

45. The culture container according to any one of claims 38 to 42, wherein the gas in the headspace comprises about 24% to about 25% CO2.

46. The culture container according to any one of claims 36 to 45, wherein the gas in the headspace comprises, comprises about, comprises at least, comprises at least about, comprises less than or comprises less than about, 20%, 25%, 26%, 27%, 28%, 28.1%, 28.3%, 28.3%, 28.4%, 28.5%, 28.6%, 28.7%, 28.8%, 28.9%, 29%, 30%, 31%, 32%, 33%, 34%, 35% or 40%, or N2 in a range defined by any two of the foregoing values.

47. The culture container according to any one of claims 38 to 45, wherein the gas in the headspace comprises about 0% to 30% N2.

48. The culture container according to any one of claims 38 to 45, wherein the gas in the headspace consists of or consists essentially of O2, CO2 and N2.

49. The culture container according to any one of claims 27 to 48, wherein the microbial growth is the growth of a microorganism selected from the following: Atopobium defectivum, Acinetobacter lwoffii, Aggregatibacter actinomycetemcomitans, Aerococcus viridans, Alcaligenes faecalis, Bacillus subtilis, Candida albicans, Candida glabrata, Cardiobacterium hominis, Corynebacterium jeikeium, Cryptococcus neoformans, Eikenella corrodens, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Granulicatella adiacens, Haemophilus influenzae, Haemophilus influenzae, Haemophilus influenzae type a, Haemophilus influenzae type b, Haemophilus parainfluenzae, Kingella kingae, Klebsiella pneumoniae, Leuconostoc species, Micrococcus luteus, Neisseria gonorrhoeae, Neisseria meningitidis, Pediococcus acidilactici, Proteus mirabilis, Providencia stuartii, Pseudomonas aeruginosa, Rothia mucilaginosa, Saccharomyces cerevisiae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus sanguinis.

50. A system for detecting the presence of a microorganism in a sample, the system comprising: The culture container according to any one of claims 27 to 49; A detector for obtaining a signal from a sensor; A computer configured to determine whether the signal obtained by the detector indicates the presence of microorganisms in the microbial growth medium.

51. A method for culturing microorganisms in a culture sample, the method comprising: Inoculating the microbial growth medium according to any one of the preceding claims with the sample, and culturing the microorganisms in the sample in the microbial growth medium.

52. The method according to claim 51, wherein the method further comprises detecting the presence of microorganisms in the sample.

53. The method according to claim 51 or 52, wherein the inoculation step comprises adding the sample to the microbial growth medium in the culture container according to any one of claims 27 to 50.

54. The method according to claim 53, wherein detecting the presence of the microorganisms in the sample comprises monitoring a signal in the sensor indicating the growth of the microorganisms in the microbial growth medium.

55. The method according to claim 54, wherein the signal indicates a change in the pH of the microbial growth medium.

56. The method according to claim 54, wherein the signal indicates a change in CO2 in the gas in the headspace of the culture container.

57. The method according to any one of claims 52 to 55, wherein the microorganism is Candida, and the TTD of Candida is shortened compared to culturing Candida in the same microbial growth medium, wherein the amount of iron in the same microbial growth medium is less than about 0.4 ppm and the amount of copper is less than about 0.04 ppm, or wherein there is no iron and copper in the same microbial growth medium.

58. The method according to any one of claims 51 to 56, wherein the culturing comprises maintaining the microbial medium at a temperature of 35°C to 39°C or 37°C.

59. The method according to any one of claims 51 to 58, wherein the method is performed using the system according to claim 50.

60. The method according to any one of claims 51 to 59, wherein the sample is selected from: biological samples such as blood, serum, plasma, urine, cerebrospinal fluid, pleural effusion, thoracic fluid, thoracentesis fluid, peritoneal fluid, ascites, pericardial fluid, bone marrow, synovial fluid; or industrial samples such as food or drug ingredients.

61. The culture container according to any one of claims 51 to 60, wherein the sample is a biological sample from a patient positive for systemic inflammatory response syndrome (SIRS) or a septic patient.

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