The following points highlight the top nine types of common laboratory media. The types are: 1. Peptone Water 2. Nutrient Broth 3. Glucose Broth 4. Sugar Media 5. Urea Medium 6. Glycerol Saline Transport Medium 7. Cary Blair Medium 8. Stuart’s Transport Medium 9. Venkatraman-Ramakrishnan (VR) Medium.
Common Laboratory Media: Type # 1. Peptone Water:
Contains 1% peptone and 0.5% sodium chloride dissolved in water. It does not contain meat extract. The medium is colourless. Peptone water is used to study proteolytic activity of bacteria viz; formation of indole and hydrogen sulphide (H2S). Motility of bacteria is best studied from peptone water culture; peptone water is used as a base for sugar media.
Common Laboratory Media: Type # 2. Nutrient Broth:
It is a pale yellow coloured liquid. The first step is to prepare the meat extract. This is done by extracting meat in cold with water. It is then heated to coagulate the dissolved proteins and filtered. The filtrate is the required meat extract.
Peptone and sodium chloride are added to the above for preparation of nutrient broth. It is sterilised by autoclaving. It is a common fluid medium for non-fastidious organisms and serves as a base for preparation of solid media.
Common Laboratory Media: Type # 3. Glucose Broth:
It is a pale yellow liquid which contains basically nutrient broth to which one per cent glucose is added. It is mainly used to grow anaerobic streptococci. Hartley broth contains lean meat, pancreatic extract, is yellow transparent and is used for blood culture.
Common Laboratory Media: Type # 4. Sugar Media:
Various carbohydrates, alcohols etc. (designated as ‘SUGAR’) are fermented by bacteria with the formation of acid or acid and gas. Sugar media are used to study these fermentation reactions. Appropriate sugars are added to peptone water in the proportion of one per cent.
A suitable indicator is also incorporated to detect the formation of acid. Usually Andrade’s indicator is used which is colourless in alkaline and neutral pH and turns pink in presence of acid. To detect the production of gas, a small tube (Durham tube) is placed in an inverted position in the medium. The air is replaced by fluid during sterilisation by heat.
Gas, if produced, is seen as a bubble at the top of the Durham tube. The sugar media are sterilised in a steam sterilizer at 100°C for 20 minutes on three successive days. The common sugar media are Glucose, Sucrose, Lactose, Mannitol and Dulcitol. Note the coloured plugs to differentiate them (Glucose, blue; Sucrose, white; Mannitol, pink; lactose, red; Dulcitol, yellow or coloured beads).
Common Laboratory Media: Type # 5. Urea Medium:
It contains peptone, sodium chloride, glucose, buffer, 20% urea, phenol red, pH 6.8-6.9. After inoculation heavily with cultures and incubation at 37°C for 4 hrs., and then overnight at room temperature, urease at room temperature positive cultures produce purple pink colour due to a change in the colour of the indicator. Thus, urease producing organisms are identified.
Common Laboratory Media: Type # 6. Glycerol Saline Transport Medium:
This medium contains glycerol, sodium chloride, buffer, phenol red, water. It is used to transport enteric bacilli to the laboratory. This fluid should not be used if it becomes acid, indicated by a change in colour to yellow (Fig. 5.2(a)).
Common Laboratory Media: Type # 7. Cary Blair Medium:
This is a buffered solution of sodium chloride, sodium thioglycollate, disodium phosphate and calcium chloride at pH 8.4. It is a suitable transport medium for Vibrio cholera. At pH 7.4, it can be used to transport Salmonella, Shigella and Campylobacter (Fig. 5.2(b)).
Common Laboratory Media: Type # 8. Stuart’s Transport Medium:
It is composed of anaerobic salt solution, methylene blue, agar 0.6% pH 7.3-7.4. This is soft agar medium used to maintain the viability of gonococci on swabs during transport to the laboratory (Fig. 5.2(c)).
Common Laboratory Media: Type # 9. Venkatraman-Ramakrishnan (VR) Medium:
A simple modified form of this medium is prepared by dissolving 20 g crude sea salt and 5 g Peptone in one litre of distilled water and adjusting the pH to 8.6-8.8. It is dispensed in screw-capped bottles in 10-15 ml amounts. About 1-3 ml stool is to be added to each bottle. In this medium vibrio’s do not multiply, but remain viable for several weeks (Fig. 5.2(d)).
a. Nutrient agar:
It is prepared by adding agar to nutrient broth in proportion of 2 to 3 percent. The broth is put in the steamer to dissolve the agar. The agar broth fluid is filtered hot if necessary and autoclaved. It becomes solid when cooled. The medium is almost transparent. It is a common- purpose solid medium for growth of non-fastidious organisms, further, it serves as a base for the ‘Enriched media’ (Fig. 5.4).
b. Blood agar:
It is prepared by adding defibrinated or citrated blood to nutrient agar in the proportion of 5 to 10 per cent. Blood is collected aseptically and added to the already sterilised nutrient agar which has been melted and cooled to about 55 C. The medium is used for the cultivation of fastidious organisms which fail to grow on nutrient. Agar (Fig. 5.5).
c. Chocolate agar:
It is prepared by heating 10% sterile blood in sterile nutrient agar. Melt the agar, cool it in a water bath at 75 C, add the blood & allow the medium to remain at 75 C, mixing the blood & agar by gentle agitation from time to time until the blood becomes chocolate-brown in colour, within 10 min. Then pour as slopes or plates. This medium is used for the cultivation of gonococci and meningococci which form greyish colonies.
d. Tellurite chocolate agar:
It is heated blood agar to which potassium tellurite (0.4%) has been added. It inhibits the growth of many commensal organisms likely to be present in the throat but not the diphtheria bacilli and related organisms. Moreover, these latter organisms form grey or black colonies on the medium as they reduce potassium tellurite. The medium is opaque brown in appearance.
e. Loeffler’s serum:
Consists of human or animal serum added to Glucose broth. The collection of blood and separation of serum are carried out with aseptic precautions. The medium is solidified by inspissation. The medium is opaque white in appearance. It is mostly used for cultivation of the diphtheria bacillus. It can be employed to study proteolytic activity of bacteria (liquefaction of serum) (Fig. 5.5(c)).
f. Dorset’s egg medium:
It is prepared by inspissating a mixture of whole egg fluid 3 part and saline 1 part. The medium is yellow and opaque. When glycerinated, the medium is used for cultivation of tubercle bacilli; without glycerine it is useful for the preservation of stock cultures of enteric bacilli.
g. Lowenstein jensen medium:
It contains a number of mineral salt, glycerine, starch, egg proteins and malachite green. The last is a bactericidal agent. This medium is used for the cultivation of tubercle bacilli.
h. MacConkey medium:
It is used for the isolation of intestinal pathogens. Bile salt inhibits the growth of Gram-positive cocci. Pathogenic Gram-negative enteric bacilli (Salmonella, Shigella) do not ferment lactose and form pale colonies. Escherichia coli (which are commensals), on the other hand, ferment lactose producing acid and, therefore, their colonies are pink.
The medium is thus both an inhibitory and differentiating one. A number of media are described which work on the same principle viz, D.E.C. medium of Panja and Ghosh and Leifson’s desoxycholate citrate medium etc. They differ from the basic medium described above in containing inhibitory substances other than bile salt. All appear reddish brown and transparent (Fig. 5.3).
i. Wilson and blair medium:
This medium is utilised for isolation of typhoid and paratyphoid organisms from heavily contaminated material like sewage. It contains sodium sulphate, Bismuth ammonium citrate and brilliant green. The last one inhibits Escherichia coli.
Typhoid and paratyphoid organisms form black colonies as they reduce sulphate to sulphide in presence of glucose—precipitating insoluble bismuth sulphide (which is black). It is also a differentiating and inhibitory medium.
j. Robertson’s cooked meat medium:
Minced meat is boiled in alkaline water. The softened meat is then partially dried between folds of filter paper and added to nutrient broth. The medium is autoclaved for sterilisation. The medium is used for cultivation of anaerobic organisms.
k. Litmus milk medium:
It is prepared by adding litmus solution to skimmed milk. The medium is autoclaved or boiled for sterilisation. It is used for studying proteolytic and saccharolytic activity of Clostridium welchii.
l. Thiosulphate citrate bile salt sucrose (TCBS) medium:
This medium- containing thiosulphate, citrate, bile salt, bromothymol blue and sucrose—is available commercially and is very widely used at present. Vibrio cholerae produce large yellow convex colonies which may become green on continued incubation.
m. Triple sugar iron (TSI) medium:
This medium contains glucose, lactose and sucrose, phenol red indicator (to indicate fermentation), ferrous sulphate (to demonstrate H2S, indicated by blackening of the butt). It is orange red when uninoculated. It is used to identify the entero-bacteriaceae. The glucose concentration is 1/10th of concentration of lactose and sucrose so to detect the fermentation of glucose alone.
The small amount of acid produced by fermentation of glucose is oxidised rapidly in the slant which reverts to alkaline (red); in contrast, under lower O2 tension in the butt, the acid reaction (yellow) is maintained. Free exchange of air may be permitted in the slant through the use of loose cotton plug to promote oxidation of acid produced by glucose fermentation in the slant.
This reaction should be read after 24 hrs’ incubation.
n. Simmon’s citrate medium:
It is modified Koser’s medium with agar and an indicator (bromothymol blue). It is used to test the ability of an organism to utilise citrate as the sole source of carbon and energy for growth and an ammonium salt as the sole source of nitrogen. It is also called citrate utilisation test.
Positive = Blue colour and growth of bacteria.
Negative = Original green colour and»no growth.
o. Xylose lysine desoxycholate (XLD) agar:
Yeast extract agar with lysine, xylose, lactose, sucrose and ferric ammonium citrate; sodium desoxycholate inhibits Gram- positive organisms, phenol red indicator. It is used to isolate and differentiate Salmonella and Shigella from other Gram-negative enteric bacilli.
Wilson Blair and Lowenstein Jensen media are green. Wilson Blair, however, is usually poured in Petri dishes whereas Lowenstein Jensen medium has to be kept in screw capped bottles or tubes with parafinished plugs.
It is a polysaccharide prepared from a seaweed grown on the Japanese and Chinese sea coasts. It is available in dry strands or in powder forms. It has no nutrient property and is used for solidifying the media. It does not set till it is cooled to 42°C.
Therefore blood, serum etc. can be added to nutrient agar at 55°C at which temperature, the medium is still fluid and there is no risk of denaturation of the serum proteins added. Peptone is golden granuled protein powder which has the ability to support the growth of bacteria.