Selective and Differential Bacterial Isolation Culture
Materials Required
Principle
Selective and differential bacterial isolation culture separates viable bacteria by plating specimens on solid media that both suppress unwanted organisms and display interpretable colony phenotypes. MacConkey-type media use bile salts to support intestinal Gram-negative organisms while distinguishing lactose-fermenting bacteria in fecal material; sorbitol-MacConkey replaces lactose with sorbitol to detect non-sorbitol-fermenting Escherichia coli O157:H7; mannitol salt agar selectively recovers Staphylococcus aureus and differentiates yellow mannitol-positive colonies; chromogenic orientation media use enzyme-substrate color reactions to presumptively distinguish uropathogens on primary plates[1][2][3][4][5][6].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use the selective/differential agar matched to the isolation goal: MacConkey agar for enteric Gram-negative lactose-fermentation screening, sorbitol-MacConkey agar for suspected E.coli O157:H7, mannitol salt agar for S.
• Aureus recovery, and CHROMagar Orientation for urine-pathogen differentiation[1][2][3][4][5][6].
• The supported detection chemistries are medium-embedded indicators or substrates: lactose/sorbitol fermentation readouts in MacConkey-type media, mannitol fermentation readout in mannitol salt agar, and chromogenic enzyme substrates in CHROMagar Orientation[1][2][3][4][5][6].
• Use sterile Petri dishes containing the selected agar, sterile inoculating loops including calibrated urine loops when quantitative urine culture is performed, an aerobic incubator set to 35 ± 2°C or 35°C as reported, and standard colony-reading tools for visual assessment of colony count, morphology, pigmentation, and mixed growth[4][5][6].
• Aureus recovery, and CHROMagar Orientation for urine-pathogen differentiation[1][2][3][4][5][6].
• The supported detection chemistries are medium-embedded indicators or substrates: lactose/sorbitol fermentation readouts in MacConkey-type media, mannitol fermentation readout in mannitol salt agar, and chromogenic enzyme substrates in CHROMagar Orientation[1][2][3][4][5][6].
• Use sterile Petri dishes containing the selected agar, sterile inoculating loops including calibrated urine loops when quantitative urine culture is performed, an aerobic incubator set to 35 ± 2°C or 35°C as reported, and standard colony-reading tools for visual assessment of colony count, morphology, pigmentation, and mixed growth[4][5][6].
Experimental Procedure
• Prepare or obtain agar plates corresponding to the study question and sample type.
• In the CHROMagar Orientation urine study, plates were poured in 90-mm dishes, stored at 4-6°C protected from light, and used within 10 weeks; quality control included sterility, culture response to a low inoculum, and expected chromogenic reactions using ATCC strains[5].
• For urine culture on CHROMagar Orientation, inoculate urine in parallel or as a primary plate using a calibrated loop.
• Published studies used a 10-µl loop with aerobic incubation at 35 ± 2°C overnight or up to 48 h on weekends, and another routine-laboratory comparison used a 0.001 calibrated loop with incubation in air at 35°C overnight, with interpretation difficult before 16 h[5][6].
• Select the medium according to the target organism or specimen.
• Use MacConkey-type medium for Gram-negative enteric differentiation, sorbitol-MacConkey for suspected E. coli O157:H7, mannitol salt agar for S. aureus, and CHROMagar Orientation for urine specimens requiring direct differentiation of common uropathogens[1][2][3][4][5][6].
• Mix the specimen and streak the plate to obtain isolated colonies.
• For urine, published CHROMagar Orientation studies used calibrated-loop inoculation to allow colony-count interpretation, and routine comparison studies used the same streaking pattern across blood agar, MacConkey agar, and CHROMagar Orientation[5][6].
• Incubate plates aerobically.
• CHROMagar Orientation urine-culture studies reported incubation at 35 ± 2°C overnight or 48 h on weekends, and another study reported incubation in air at 35°C for 10-24 h, with CO plates difficult to interpret before 16 h[5][6].
• Read plates for growth, colony count, colony morphology, and color.
• On CHROMagar Orientation, color and morphology supported direct differentiation of E. coli, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Acinetobacter spp., Enterococcus spp., staphylococci, streptococci, and yeasts in urine-culture studies, but Klebsiella-Enterobacter-Citrobacter isolates produced similar pigmentation and required additional identification[5][6].
• Use confirmatory testing when the primary plate result is presumptive or ambiguous.
• CHROMagar Orientation studies used biochemical reactions, Gram stain, coagulase testing for suspected S. aureus, indole testing for differentiation of P. mirabilis and Morganella morganii, and additional tests when colony color could not resolve related groups[5][6].
• For S. aureus on mannitol salt agar, yellow colonies at more than 1+ quantity were reported as S. aureus in a cystic-fibrosis specimen study, and antimicrobial susceptibility testing from mannitol salt agar isolates correlated with testing from blood agar isolates[3].
• For suspected E. coli O157:H7, interpret sorbitol-MacConkey as a differential screen because E. coli O157:H7 did not ferment sorbitol in the original evaluation, and the authors recommended routine SMAC stool culture for detection of that serotype in hemorrhagic-colitis investigations[4].
• Record whether growth is present, whether colony morphology suggests one organism or mixed flora, the colony count when a calibrated loop is used, and the differential color phenotype specified for the chosen medium.
• In CHROMagar Orientation urine studies, results were compared with blood agar and MacConkey agar, and interpretation included detection of no growth, low-count growth, mixed growth, single pathogens, and mixed clinically significant pathogens[5][6].
• Use positive control strains that are expected to grow and produce the correct differential phenotype.
• One CHROMagar Orientation evaluation used S. aureus ATCC 25923, E. faecalis ATCC 19433, E. coli ATCC 25922, P. mirabilis ATCC 4630, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 13883, and Candida albicans ATCC 10231 for quality control[5].
• Interpret primary culture as presumptive unless the cited study showed that direct reporting was acceptable for that target and condition.
• CHROMagar Orientation allowed presumptive identification of several uropathogens but required extra tests for Klebsiella-Enterobacter-Citrobacter grouping and some streptococci, while mannitol salt agar supported direct reporting of yellow S. aureus colonies only under the study’s stated quantity criterion[3][5][6].
• In the CHROMagar Orientation urine study, plates were poured in 90-mm dishes, stored at 4-6°C protected from light, and used within 10 weeks; quality control included sterility, culture response to a low inoculum, and expected chromogenic reactions using ATCC strains[5].
• For urine culture on CHROMagar Orientation, inoculate urine in parallel or as a primary plate using a calibrated loop.
• Published studies used a 10-µl loop with aerobic incubation at 35 ± 2°C overnight or up to 48 h on weekends, and another routine-laboratory comparison used a 0.001 calibrated loop with incubation in air at 35°C overnight, with interpretation difficult before 16 h[5][6].
• Select the medium according to the target organism or specimen.
• Use MacConkey-type medium for Gram-negative enteric differentiation, sorbitol-MacConkey for suspected E. coli O157:H7, mannitol salt agar for S. aureus, and CHROMagar Orientation for urine specimens requiring direct differentiation of common uropathogens[1][2][3][4][5][6].
• Mix the specimen and streak the plate to obtain isolated colonies.
• For urine, published CHROMagar Orientation studies used calibrated-loop inoculation to allow colony-count interpretation, and routine comparison studies used the same streaking pattern across blood agar, MacConkey agar, and CHROMagar Orientation[5][6].
• Incubate plates aerobically.
• CHROMagar Orientation urine-culture studies reported incubation at 35 ± 2°C overnight or 48 h on weekends, and another study reported incubation in air at 35°C for 10-24 h, with CO plates difficult to interpret before 16 h[5][6].
• Read plates for growth, colony count, colony morphology, and color.
• On CHROMagar Orientation, color and morphology supported direct differentiation of E. coli, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Acinetobacter spp., Enterococcus spp., staphylococci, streptococci, and yeasts in urine-culture studies, but Klebsiella-Enterobacter-Citrobacter isolates produced similar pigmentation and required additional identification[5][6].
• Use confirmatory testing when the primary plate result is presumptive or ambiguous.
• CHROMagar Orientation studies used biochemical reactions, Gram stain, coagulase testing for suspected S. aureus, indole testing for differentiation of P. mirabilis and Morganella morganii, and additional tests when colony color could not resolve related groups[5][6].
• For S. aureus on mannitol salt agar, yellow colonies at more than 1+ quantity were reported as S. aureus in a cystic-fibrosis specimen study, and antimicrobial susceptibility testing from mannitol salt agar isolates correlated with testing from blood agar isolates[3].
• For suspected E. coli O157:H7, interpret sorbitol-MacConkey as a differential screen because E. coli O157:H7 did not ferment sorbitol in the original evaluation, and the authors recommended routine SMAC stool culture for detection of that serotype in hemorrhagic-colitis investigations[4].
• Record whether growth is present, whether colony morphology suggests one organism or mixed flora, the colony count when a calibrated loop is used, and the differential color phenotype specified for the chosen medium.
• In CHROMagar Orientation urine studies, results were compared with blood agar and MacConkey agar, and interpretation included detection of no growth, low-count growth, mixed growth, single pathogens, and mixed clinically significant pathogens[5][6].
• Use positive control strains that are expected to grow and produce the correct differential phenotype.
• One CHROMagar Orientation evaluation used S. aureus ATCC 25923, E. faecalis ATCC 19433, E. coli ATCC 25922, P. mirabilis ATCC 4630, P. aeruginosa ATCC 27853, K. pneumoniae ATCC 13883, and Candida albicans ATCC 10231 for quality control[5].
• Interpret primary culture as presumptive unless the cited study showed that direct reporting was acceptable for that target and condition.
• CHROMagar Orientation allowed presumptive identification of several uropathogens but required extra tests for Klebsiella-Enterobacter-Citrobacter grouping and some streptococci, while mannitol salt agar supported direct reporting of yellow S. aureus colonies only under the study’s stated quantity criterion[3][5][6].
Troubleshooting
CHROMagar Orientation color suggests Klebsiella, Enterobacter, or Citrobacter but species-level distinction is unclear.
Possible Cause:These isolates produced similar pigmentation on CHROMagar Orientation.
Literature-supported Solution:
Report as a coliform group only if appropriate for the laboratory workflow and perform additional biochemical or identification testing for final identification[5][6].
CHROMagar Orientation plate is hard to interpret early.
Possible Cause:One routine-laboratory study reported that plates were difficult to interpret when read before 16 h
Literature-supported Solution:
Read after overnight aerobic incubation rather than before 16 h[6].
Urine-culture colony counts or organism recovery differ between media.
Possible Cause:A bench comparison found that inconsistent results could be explained partly by inoculum effects and improper urine inoculation technique
Literature-supported Solution:
Supervisors should review technique and directly observe setup-protocol proficiency throughout the year[6].
CHROMagar Orientation suggests group B streptococci or lactobacilli but visual distinction is unreliable.
Possible Cause:Direct visual differentiation of group B streptococci from lactobacilli was not possible in one study
Literature-supported Solution:
Use Gram stain morphology to recognize lactobacilli and apply additional identification methods when needed[6].
References:
- [1]. Macconkey A. Lactose-Fermenting Bacteria in Faeces. J Hyg (Lond). 1905;5(3):333-379. [Content Brief]
- [2]. Macconkey AT. Bile Salt Media and their advantages in some Bacteriological Examinations. J Hyg (Lond). 1908;8(3):322-334. [Content Brief]
- [3]. Sharp SE, et al. Comparison of mannitol salt agar and blood agar plates for identification and susceptibility testing of Staphylococcus aureus in specimens from cystic fibrosis patients. J Clin Microbiol. 2006;44(12):4545-4546. [Content Brief]
- [4]. March SB, et al. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol. 1986;23(5):869-872. [Content Brief]
- [5]. Samra Z, et al. Evaluation of use of a new chromogenic agar in detection of urinary tract pathogens. J Clin Microbiol. 1998;36(4):990-994. [Content Brief]
- [6]. D'Souza HA, et al. Practical bench comparison of BBL CHROMagar Orientation and standard two-plate media for urine cultures. J Clin Microbiol. 2004;42(1):60-64. [Content Brief]
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