Carbohydrate Fermentation by Different Microbes

 Aim

To determine the specific carbohydrate fermentation profiles of different bacteria.

Principle:

Microorganisms metabolize carbohydrates in various ways. Fermentation is an anaerobic process where microbes break down carbohydrates, typically producing acid and/or gas as byproducts. The carbohydrate fermentation test detects acid production as indicated by a change in the pH indicator (e.g., phenol red) in the medium from its initial red/orange colour to yellow. Gas production is indicated by the presence of gas bubbles collected in an inverted Durham tube placed inside the fermentation broth.

Materials Required

Bacterial cultures such as Staphylococcus aureus, Escherichia coli, Pseudomonas and Bacillus, Basal fermentation broth containing carbohydrates such as Glucose, Sucrose, Maltose and Lactose, other routine microbiological facilities.

Procedure

Procedure

1.  Basal fermentation broth containing carbohydrates such as glucose, sucrose, maltose and lactose were prepared

2.   The broth was dispensed into test tubes with Durham’s tube and sterilized.

3. Using a sterile inoculating loop, each labelled fermentation tube (glucose, sucrose, maltose and lactose) was aseptically inoculated with a small amount of the specific bacterial culture. A negative control was set up for each sugar without bacterial inoculation

4. All inoculated tubes were incubated at 37°C for 24 - 48 hours.

      5. The tubes were observed for colour change and gas production.

 Observation and result

Acid production is indicated if the medium turns yellow (from red/orange) and Gas production is indicated if bubbles are visible in the inverted Durham tube.

	Staphylococcus aureus	Escherichia coli	Pseudomonas	Bacillus
Glucose
Sucrose
Maltose
Lactose

         A = acid only (broth turned yellow)

       AG = acid and gas (broth turned yellow + bubble trapped in Durham tube)

Composition of Carbohydrate Broth

Peptone- 10.00 gm

Beef extract- 1.00 gm

Sodium Chloride- 5.00 gm

Glucose/Sucrose/Maltose/Lactose - 10 gm

Phenol Red- 0.018 gm

Distilled water – 1000 ml

Mueller Hinton Agar

Mueller and Hinton developed Mueller Hinton Agar (MHA) in 1941 for the isolation of pathogenic Neisseria species. It is now commonly used for the routine susceptibility testing of non-fastidious microorganism by the Kirby-Bauer disk diffusion technique.

MHA is recommended for the diffusion of antimicrobial agents impregnated on paper disc through an agar gel as described in CLSI Approved Standard. Zone diameters are established for each antimicrobial agent determining resistant, intermediate, and sensitive results for pathogenic microorganisms

Mueller Hinton Agar with 5% sheep blood and Mueller Hinton Agar with Hemoglobin have been recommended for antimicrobial susceptibility testing of Streptococcus pneumoniae and Haemophilus influenza.

MHA

Mueller Hinton Media contains Beef Extract, Acid Hydrolysate of Casein, Starch and Agar. Beef Extract and Acid Hydrolysate of Casein provide nitrogen, vitamins, carbon, amino acids, sulphur and other essential nutrients. Starch is added to absorb any toxic metabolites produced. Starch hydrolysis yields dextrose, which serves as a source of energy. Agar is the solidifying agent. Final pH 7.3 ± 0.1 at 25ºC

Composition of Mueller Hinton Agar (MHA)

Ingredients	Gms/Litre
Beef extract	2.0
Acid hydrolysate of casein	17.5
Starch	1.5
Agar	17.0

 Uses of MHA

1. The major use of Mueller Hinton Agar is for antimicrobial susceptibility testing. It has become the standard medium for the  Kirby-Bauer method

2. It can be used to cultivate Neisseria

3. It is specified in FDA Bacteriological Analytical Manual for food testing, and procedures commonly performed on aerobic and facultative anaerobic bacteria.

Why MHA is used for antibiotic susceptibility testing?

1. It is a non-selective, non-differential medium. This means that almost all organisms plated on here will grow.

2. It contains starch. Starch is known to absorb toxins released from bacteria, so that they cannot interfere with the antibiotics. It also mediates the rate of diffusion of the antibiotics through the agar.

3. It is a loose agar. This allows for better diffusion of the antibiotics than most other plates. A better diffusion leads to a truer zone of inhibition.

4. MHA shows acceptable batch-to-batch reproducibility for susceptibility testing.

5. MHA is low in sulfonamide, trimethoprim, and tetracycline inhibitors (i.e. concentration of inhibitors thymidine and thymine is low in MHA).

6. It supports the growth of most non-fastidious bacterial pathogens andiv.

Limitations of MHA

1. Numerous factors can affect results: inoculum size, rate of growth, medium formulation and pH. Strict adherence to protocol is required to ensure reliable results.

2. Drug inactivation may result from the prolonged incubation times required by slow growers.

3. This medium is recommended for susceptibility testing of pure cultures only. Inoculum density may affect the zone size.

4. Heavy inoculum may result in smaller zones or too less inoculum may result in bigger zones. 

5. Fastidious organisms may not grow on this medium and may require supplementation of blood. 

6. As antimicrobial susceptibility is carried with antibiotic disc, proper storage of the disc is desired which may affect the potency of the disc. 

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References

l Atlas R.M and Snyder J.W. 2014. Handbook of Media for Clinical and Public Health Microbiology. CRC Press. Taylor & Francis Group. 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742. Page no.324-325

l http://himedialabs.com/TD/M173.pdf

l https://microbiologyinfo.com/mueller-hinton-agar-mha-composition-principle-uses-and-preparation/

l https://microbeonline.com/why-mueller-hinton-agar-is-used-in-routine-antibiotic-susceptibility-testing