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The following points highlight the three types of cultural medium used in clinical microbiology. The types are: 1. Un-Inoculated Culture Medium 2. Inoculated Culture Medium Showing Colonies 3. Medium for Biochemical Identification Test .
1. Un-Inoculated Culture Medium:
(A) Basal Medium:
(l) Peptone water (Fig. 10.1) — almost colourless liquid, dispensed in 4″ x ½” tube, properly cotton plugged. Composition and sterilisation.
Uses:
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(i) Basal medium for preparation of nutrient broth and sugar fermentation medium.
(ii) Many non-fastidious organism is grown in this medium for preparation of inoculum for anti-bio-gram testing.
(iii) It is the medium for indole test.
(2) Nutrient broth (Fig. 10.2) — faint straw coloured liquid dispensed in 4″ x ½” tube.
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Uses:
(i) Bacteria is grown in nutrient broth for motility test.
(ii) Basal medium for preparation of nutrient agar.
(3) Nutrient agar (Fig. 10.3) — Faint straw coloured/colourless solid medium in 45 mm radius petri- plates or slants. Its composition and sterilisation.
Uses:
(i) For growth of organism for pigment production, serological test, bacteriocin typing.
(ii) To note the colony character.
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(iii) Basal medium for preparation of blood agar.
(b) Enriched Medium:
(1) Blood agar (Fig. 10.4) —Red, opaque solid medium in 45 mm radius petriplates, slants in test tubes.
Uses:
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(i) Primary culture of throat swab, sputum, pus and other body fluids.
(ii) To see haemolytic property of streptococcus.
(iii) To perform CAMP test.
(iv) To see swarming property of Proteus and C. tetani.
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(2) Chocolate agar (Fig. 10.5) — Chocolate coloured opaque solid medium in 45 mm petriplates.
Composition and sterilisation is same as ‘Blood agar’. Before medium is poured, it is heated at about 80°C in water bath till blood starts lysing indicated by changing colour from red towards chocolate. Overheating destroys the medium. Lysis of RBC, liberates X-factor in the medium.
Uses:
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(i) Primary culture of sputum for growth of H. influenza.
(ii) Primary culture of urethral and endocervical swab for growth of Neisseria gonorrhoeae. To avoid contaminants vancomycin, colistin and nystatin is added to the medium.
(iii) Primary culture of CSF to grow N. meningitidis and H. influenzae. For the latter ‘V’ factor disc is placed at the primary inoculum site.
(3) Lowenstein-Jensen (LJ) medium (Fig. 10.6) Light green solid medium dispensed in screw cap bottle. Composition and sterilisation.
Uses:
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(i) To culture M. tuberculosis.
(ii) To perform anti-tubercular drug sensitivity test by absolute concentration method.
Some points to remember:
1. Malachite green, imparting green colour to the medium, is inhibitory for growth of contaminants.
2. Egg fluid, on inspissation, solidifies the medium. It is the prime nutrient of the medium.
(4) Dorset egg medium (Fig. 10.7):
Medium is like a hard-boiled egg fluid, dispensed in screw cap bottles (McCartney or Bijou bottle).
Sterilisation:
Inspissation.
Uses:
(i) Culture of C. diphtheriae.
(ii) Preservation of bacterial isolates. Culture is done on slope. Once colony appears, 10% Glycerol-saline (sterile) is added to cover the colonies.
(5) Robertson’s Cooked Meat (RCM) medium (Fig. 10.8):
Autoclaved meat particle, covering 1/2 of the container, in nutrient broth. Broth level is about 1/2″ above the meat level. In some instances, there is a covering layer of paraffin too. Medium is dispensed in MacCartney’s bottle.
Sterilisation:
Autoclaving (15 Lb for 20 minutes).
(6) Blood culture bottle with medium (Fig. 10.9):
About 50 ml straw coloured fluid in blood culture bottle. Capacity of the bottle is about 100 ml. It is screw capped. Cap is provided with a rubber washer and a fenestration for introduction of needle of a syringe.
Composition:
Brain Heart Infusion Broth/Trypti case Soya Broth/Brucella Broth/Hartley’s broth/1% Glucose broth/0.5% Bile Broth.
Sterilisation:
Autoclaving (15 Lb for 20 minutes).
Use:
Blood culture in bacteremia and septicaemic conditions (e.g. Enteric fever, Bacterial endocarditis, Lobar pneumonia, Meningococcal meningitis etc.).
[In Castaneda medium, liquid blood culture broth and solid nutrient agar is incorporated in blood culture bottle in the form of diphasic medium (Fig. 10.10)].
Advantage:
No regular sub-culture is required, as is done in monophasic medium, to note growth of organism. Solid slant provided will show growth of colonies. The inoculated bottle is tilted once daily to facilitate inoculation on solid slant.
7. Loeffler’s serum slope (Fig. 10.11):
White solid slant dispensed in test tube or screw cap bottles.
Composition:
Nutrient broth with serum.
Sterilisation:
Inspissation.
Uses:
(i) Rapid culture for C. diphtheriae for morphology study in 6-8 hours.
(ii) With addition of sugar and indicator (phenol red), may be used to study fermentation property of Neisseria.
(c) Selective and Indicator Medium:
(1) MacConkey’s agar (Fig. 10.12):
Red, opaque solid medium in 45 mm radius petriplates, slants in test tubes. Composition and sterilisation.
Uses:
(i) Because of presence of bile salt, it inhibits growth of Staphylococcus, Micrococcus, Bacillus, E. coli and Klebsiella. So it is used for isolation of salmonella and shigella from stool.
(ii) Although it is not an ideal medium, still it is commonly used for urine culture.
(iii) It is used to isolate Enterobacteriaceae, Pseudomonadaceae, A.G.N.Rs, Group D streptococcus from samples, likely to be contaminated with gram +ve environmental bacteria and skin flora.
(2) T.C.B.S. agar (Fig. 10.13):
Green solid medium in petri plates.
Use:
It is a highly selective medium for Vibrio. So it is used to isolated Vibrio from stool sample. Moreover, the media contains sucrose and bromothymol blue indicators, helping differentiation between sucrose fermenting (e.g. V. cholerae) and non-fermenting (e.g., V. parahaemolyticus) vibrios.
[1. It is to be noted, unless procured from a well known producer of U.S., the desired result may not be obtained.
2. Sometimes bystanders like Pseudomonas, Klebsiella and Proteus grows on this medium].
(3) S.S. Agar (Fig.10.14):
Pink coloured medium, semitransparent, dispensed in petri plates.
Use:
(1) It is a highly selective medium for isolation of Salmonella, Shigella, Aero-monas and Plesiomonas from stool.
(2) It is an indicator medium too and able to differentiate between lactose fermenters (E. coli, Klebsiella) from non-Lactose Fermenters (Salmonella, Shigella, Aeromonas, Plesiomonas). H2S producers (Salmonella) produce black center colonies.
[It is to be mentioned that although inhibitory it cannot completely suppress the growth of Klebsiella, Citrobacter, Enterobacter, Proteus, Providence and E. coli].
(d) Enrichment Medium:
(1) Selenite F broth:
Brick coloured liquid medium dispensed in test tubes.
Composition:
Follow the manufacturer’s instruction.
Sterilisation:
Autoclave.
Use:
The medium is used for enrichment of the number of Salmonella, Aero monas and Plesiomonas in stool specimen. Sub-culture is done after 18 hours of incubation on S.S. Agar.
(2) Alkaline Peptone water:
10 ml transparent colourless liquid dispensed in 6’’ x 1/2″ test tubes.
Composition:
Peptone 0.5%, NaCl 2%, D.W – as required. pH – 8.4.
Sterilisation:
Autoclave.
Use:
Enrichment of vibrio from stool sample. 1 gm. amount of stool is inoculated in 10 ml medium.
2. Inoculated Culture Medium Showing Colonies:
1. Beta Haemolytic Colony on Blood Agar (Fig. 10.15A&B):
Clear zone of haemolysis with definite margin around pin point transparent colonies.
Probable diagnosis:
Pyogenic Streptococcus.
Further tests for identification:
(i) Smear examination after gram stain to show gram +ve cocci in long chain, morphologic of Streptococcus pyogenes.
(ii) Catalase test — A negative catalase test establish the genus level diagnosis of Streptococcus.
(iii) Bacitracin sensitivity test — S. pyogenes is sensitive.
(iv) CAMP Test — Positive in S. agalactiae.
(v) Carbohydrate Ag extraction followed by sensitized latex agglutination (latex particles coated with Ab against the carbohydrate Ag) test to identify the Lancefield group A, B, C & G.
2. On Blood Agar or Nutrient Agar (Fig. 10.16):
2-3 mm diameter, dome shaped, smooth surfaced, entire margined, creamy, golden yellow pigmented colonies. Pigment is restricted to the colonies.
Probable diagnosis:
Staphylococcus aureus.
Further tests for identification:
(i) Gram stained smear examination to show gram positive cocci arranged in grape like irregular cluster.
(ii) Catalase test — positive result differentiate Staphylococcus from Streptococcus.
(iii) Coagulase test — positive coagulase test confirm the presence of Staphylococcus aureus.
(iv) Phosphatase test — if coagulase test is found negative, phosphatase test clinch the species diagnosis ‘aureus’.
(v) Growth on 15% NaCl Agar – confirm genus level diagnosis of Staphylococcus when in doubt.
3. Swarming Growth on Blood Agar or Nutrient Agar (Fig. 10.17):
Spreading growth from original colonies in the form of waves covering the plate.
Probable diagnosis:
Growth of Proteus mirabilis or Proteus vulgaris.
Further tests for identification:
(i) Gram stained smear examination — Gram -ve short slender rod, arranged discretely.
(ii) Motility test — Motile bacteria.
(iii) PPA and urease test — Both positive by Proteus.
(iv) Indole test — P. mirabilis -ve, P. vulgaris +ve.
(v) H2S production — Both P. mirabilis and vulgaris +ve.
4. Nutrient Agar Plate Showing Irregular Margined Colonies with Greenish Pigment Diffusing into the Medium (Fig. 10.18):
Probable diagnosis:
Pseudomonas aeruginosa.
Further tests for diagnosis:
(i) Gram stained smear examination — short slender rods arranged discretely.
(ii) Motility test — Motile
(iii) Oxidase test — Positive.
5. MacConkey’s Agar Plate Showing Non-Mucoid (Fig.10.19):
Pink lactose fermenting, moderate size, convex, smooth/matt surfaced, entire margined, opaque colonies.
Probable diagnosis:
E. coli.
Further tests:
(i) Gram stained smear examination — Stout long rods arranged discretely.
(ii) Motility test — Motile rods.
(iii) Indole production +ve, methyl red test +ve, V.P. test -ve, Simmon citrate medium — no growth, H2S in TSI -ve.
6. MacConkey’s Agar Plate Showing Mucoid, Pink (LF) (Fig. 10.20):
Large, convex, smooth surfaced, entire margined colonies.
Probable diagnosis:
Klebsiella spp, or Enterobacter spp, or Hafnia spp.
Further tests:
Confirmatory test for Klebsiella pneumoniae sub-spp aero- genes: Indole -ve, M.R. -ve, VP +ve, Simmon’s citrate — growth +ve.
7. MacConkey’s Agar Showing Non-Lactose Fermenting Colonies (Fig. 10.21):
Probable diagnosis:
Any of the following genera — Salmonella, Shigella, Proteus, Providence, Morganella, NLF E. coli, Citrobacter, Aeromonas, Plesiomonas, Vibrio (if the medium is not NaCl deficient) a fermentative, gram negative rods (AGNR), Pseudomonadaceae.
Further tests for identification:
Step I:
Morphology study by gram stain and motility test. All are gram -ve slender rods. All are motile except Shigella.
Step II:
8. Nutrient Agar Plate Showing Rough Surfaced, Irregular Margined, Opaque, Non-Pigmented Colonies (Fig. 10.22):
Probable diagnosis:
Bacillus spp.
Further tests for identification:
(i) Gram stained smear examination:
Gram +ve stout rods with unstained space for spore. Depending upon the species, arrangement might be in chain, pallisade or discrete.
(ii) Motility test:
Motile, except B. anthracis.
(iii) Further test for species identification:
Position, sizes and shape of spore, gelatin hydrolysis.
Identification of pathogenic species:
(a) Bacillus anthracis:
Capsule + by ‘Polychrome Methylene Blue’ stain, Non-motile.
(b) Bacillus cereus:
Haemolytic colony on Blood Agar. Motility +, Egg yolk reaction +ve.
9. Lowenstein-Jensen (LJ) Medium Showing Buff/White/Yellow etc. Coloured Growth (Fig. 10.23A and 23B):
Probable diagnosis:
Colonies of Mycobacterium.
Further tests:
(i) Colony character:
Slow grower/Rapid grower, colour of pigment, whether scotochromogen or photo-chromogen, rough irregular colony/smooth moist small colonies.
(ii) Z. N. stained smear examination:
Acid fast bacillus.
(iii) Biochemical tests:
Niacin test, Aryl sulphatase test, Heat resistant catalase test, Nitratase test.
[Nowadays, conventional tests for identification is being replaced by ‘Gene Probe’, PCR, G.L.C., Line Probe Assay for quick and more accurate identification.]
10. S.D.A. Medium with Growth of Dermatophytes (Fig. 10.24):
Straw coloured slant in 6″ x 3/4” test tube with cottony/powdery Arial growth and reverse may show leathery growth with brown/red pigmentation.
Further test for identification:
(i) Lacto phenol cotton blue mount to show septate branching hyphae with macro-conidia. Often micro-conidia is also seen.
(ii) Slide micro-culture — followed by lacto phenol cotton blue mount to show macro-conidial pattern.
11. S.D.A. Medium with Growth of C. Albicans (Fig. 10.25):
Straw coloured slant in 6″ x 3/4″ test tube with big, whitish, creamy, smooth surfaced, regular margined colonies.
Further tests for identification:
(i) Gram stained smear — shows intensely gram +ve budding oval yeast cells.
(ii) Germ tube formation within 2 hours in serum.
(iii) Terminal smooth chlamydospore formation in slide culture in rice meal infusion agar.
3. Medium for Biochemical Identification Test (Inoculated/Uninoculated):
1. Monosaccharide Fermentation:
Monosaccharide fermentation is noted in growth in glucose peptone water with Durham tube to note gas production (Fig. 10.26).
Composition:
Peptone water with 1% Glucose with Andrade’s indicator, which turns red when pH becomes less than 6.4 due to fermentation of and acid production from glucose.
Sterilisation:
Base peptone is sterilised by autoclaving (15 Lb for 20 minutes). Then glucose and Andrade indicator is added, medium is poured in tubes, cotton plugged, followed by free steaming for 1 hour.
Result:
(i) Fermentation with gas production (red medium with gas in Durham tube) — by all entero- bacteriaceae (except Shigella, S. typhi, some species of Proteus, Providence, Serratia, Klebsiella and anaero- genic E. coli).
(ii) Fermentation without gas production — (red medium and no gas in Durham tube) — by Vibrio, Aeromonas (except hydrophila) Plesiomonas.
(iii) Red at surface only no gas production — indicates oxidative utilisation of glucose.
Example:
Pseudomonas.
(iv) No reaction — Example: A.G.N.R.
2. Disaccharide Fermentation:
Feature of the medium and results are very similar to glucose mentioned above. No Durham tube is required here, as we do not note gas production here.
Commonly used disaccharides fermentation test media are: peptone water with lactose/sucrose/maltose.
Lactose fermenters:
E coli, K. pneumoniae (sub spp pneumoniae and aerogenes), Enterobacter, Edward- siella, Hafnia (at 22°C).
Sucrose fermenters:
K. pneumoniae (sub-species pneumoniae and aerogenes), Enterobacter, Vibrio cholerae, some serotypes of E. coli.
(3) Test for Indole Production (Fig. 10.27):
Some bacteria utilizes tryptophan of peptone water medium to produce indole, which could be extracted by amyl alcohol. On addition of p-dimethyl- aminobenzaldehyde, extracted indole reacts to produce pink ring at surface (Fig. 10.27). +ve reaction: E. coli, Proteus vulgaris and others, -ve reaction: Klebsiella, Enterobacter and others.
(4) Test for Citrate Utilization (Fig. 10.28):
Some bacteria are able to utilise citrate as sole carbon source to produce the required macromolecules for its growth, survival and reproduction. Simmon’s citrate medium contain sodium-citrate as sole carbon source. It is a synthetic medium.
The test organism is inoculated on the slant. Inoculum should be as small as possible (a few bacteria only). In case of positive result colonies appear with change of pH to alkaline side turning the colour of the indicator from green to blue.
+ve:
Klebsiella aerogenes, Enterobacter, Citrobacter, Providencia rettgeri, S. typhimurium, Pseudomonas etc.
-ve:
E. coli, Shigella, Proteus (other than spp rettgeri)
5. Test for H2S Production (Fig. 10.29):
H2S production is tested in Triple Sugar Iron Agar medium. Bacteria to be tested is inoculated using a straight wire. Inoculation is done by stabbing the butt up to the bottom, followed by streaking on surface. On incubation following reactions are encountered depending on the genus, species or serovars of the bacteria.
Following are the reactions:
Slant Acid/Butt Acid (A/A):
Lactose fermenting, H2S -ve.
Slant Alkaline/Butt Acid (K/A):
Non-lactose fermenting, H2S -ve.
Slant Alkaline/Butt Acid with black deposit (K/A+):
Non-lactose fermenting, H2S +ve.
[Acid = Yellow colour, Alkaline = Red colour]
H2S +ve bacteria:
Proteus, Salmonella (except S. paratyphi A) Citrobacter freundii.
6. Phenyl Pyruvic Acid Deaminase Test (PPA) (Fig. 10.30):
It is used to test the ability of certain organisms to deaminate phenylalanine to phenyl pyruvic acid (PPA).
Nutrient agar slope containing DL-phenylalanine is inoculated with a fairly heavy inoculum of culture and incubate at 37°C for overnight period. Then a few drops of 10% ferric chloride solution is added.
Result:
(i) Positive PPA test — Green colour,
(ii) Negative PPA test — No colour change.
Examples of positive PPA test:
Proteus sp., Morganella sp. and Providencia sp.
