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International Medical School

Shah Alam Campus

Medical Microbiology
Laboratory Manual & Practical Experiments Work Record

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Practical Laboratory
Experiment (8)

1- ANTIBIOTIC SENSITIY TESTS


2- METHODS OF STUDYING MICROBES

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ANTIBIOTIC SENSITIVITY TESTS
Chemotherapeutic agents are chemical substances used in the treatment of infectious
diseases. Their mode of action is to interfere with microbial metabolism; thereby producing a
Bacteriostatic or bacteriocidal effect on the microorganisms, without producing a like effect on the
host cells.

Antibiotics are synthesized and secreted by some true bacteria, actinomycetes, and fungi. These
molecules are designed in nature to inhibit or prevent the growth of other microorganisms in the
environment. Today, antibiotics have been designed or modified based on this basic principle.
There are other synthetic drugs produced through drug research that are capable of inhibiting
bacterial growth.

Antibiotics act in two basic ways: prevention of cell wall or cell membrane construction and
interference with the process of cell protein synthesis. Because there are multiple steps in the
process of protein synthesis, antibiotics can block any step in this process and effectively eliminate
the bacteria.

From practical Lab 7; after isolation and identification of a pathogen most important step is the
antibiotic sensitivity testing. This is of immense help for the treatment of the patient in choosing
particular antibiotic.

There are different methods of doing the antibiotic sensitivity tests. In all of them a basic non-
inhibitory or sometimes enriched media (for exacting bacteria) is used.

METHODS:

1- Disc-Diffusion Technique:

This technique is less precise than the tube dilution method but more speedily performed and
allow the simultaneous tests of several drugs. Discs of filter paper with known exact quantity of
the given antibiotic are used.

a) Kirby-Bauer Technique: Discs are placed in different parts of the medium inoculated
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to give a lawn culture and incubated for 24 hours. Diameter of the zones of inhibition
for the different discs are measured and compared with the standard Kirby-Bauer chart
to read the sensitivity or resistance of the bacteria for the drugs (see Figure 1).

Figure 1: Kirby-Bauer testing; white paper discs containing antibiotics are placed on a plate of
bacteria. Circles of poor bacterial growth surround indicating susceptibility to the antibiotic.

KIRBY-BAUER TEST RESULTS

R = resistant I = intermediate S = sensitive MS = moderate sensitive

ZONE SIZE INTERPETIVE CHART FOR THE KIRBY-BAUER TEST


R = mm I = mm S = mm
ANTIMICROBIAL AGENT DISC CODE
or less range or more
Amoxicillin (Staph) AMC 19 20
Amoxicillin (other bacteria) AMC 13 14-17 18
Ampicillin (Staph) AM 28 29
Ampicillin (other bacteria) AM 11 12-13 14
Carbenicillin (Pseudomonas) CB 13 14-16 17
Carbenicillin (other bacteria) CB 17 18-22 23
Cefoxatime CTX 14 23
Cephalothin CF 14 15-17 18
Chloramphenicol C 12 13-17 18
Erythromycin E 13 14-22 23
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Gentamycin GM 12 13-14 15
Methicillin (used for Staph only) M (or DP) 9 10-13 14
Penicillin P 28 29
Streptomycin S 11 12-14 15
Sulfamethoxazole-trimethoprim SXT-TMP 10 11-15 16
Tetracycline TE 14 15-18 19

b) Strokes method: This method is the same as above except for the fact that the isolated
pathogen is compared with a standard strain of bacteria of known sensitivity and the
result read as comparison to the known strains.

Questions:

1. Based on this practical, why is there an antibiotic crisis?

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2. Why is the Kirby- Bauer test important to physicians and patients?

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3. What have your learned from your unit on Microbiology?

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2. The Dilution Technique: used to determine the minimal concentration of antimicrobial to
inhibit or kill the microorganism. This can be achieved by dilution of antimicrobial in either
agar or broth media. Antimicrobials are tested in log2 serial dilutions (two fold).

The tube test is too expensive of time, labour and materials for general purposes and it is normally
preserved for testing the sensitivity of glow growing organisms particularly M. tuberculosis.

It requires two fold dilutions of antibiotics in a suitable fluid medium, a constant volume of the
organisms are added to each tube and a control tube with no antibiotics is included in each test.
After incubation of 37°C for 24 hours the tubes are examined for turbidity. The tube with the
highest dilution showing no turbidity is the bacteriostatic concentration (minimum inhibitory
concentration). Bacterial concentration can be determined only by subculturing from those tubes
without visible growth. The highest dilution yielding no growth on subculture is the bactericidal
concentration.

METHODS OF STUDYING MICROBES

Examination of living Bacteria in unstained condition


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A. Wet cover slip preparation
B. Hanging drop preparation
C. Phase contrast microscopy (Demonstration)
D. Dark ground microscopy (Demonstration)

A. Wet coverslip preparation:

A small drop of material to be examined is taken on a clean glass slide and covered with a
coverslip without allowing air bubbles to form. Examined under low and high dry power
objectives.

Uses:

1. Examination of Urinary sediments. CSF. Other body fluids, etc. for puscells, crystals, casts
and bacteria.
2. Examination of vaginal secretion for presence of motile protozoan parasites like,
Trichomonas vaginalis.
3. Examination of faeces in saline and iodine preparation for helminthic ova or protozoal
cysts.

B. Hanging drop preparation:

For the study of morphology and motility of bacteria.

REQUIREMENTS:

1. A clean depression slide with paraffin ring.


2. Clean coverslip.
3. Bacterial suspension (Young broth culture).
4. Bacterial loop.
5. Burner

TECHNIQUE:

1. With sterile precaution transfer a small drop of suspension on to the centre of a clean cover
slip.
2. Pass the paraffin ring slide over the flame once or twice.
3. Invert the slide with paraffin ring over the cover slip so that the drop of culture is in the
center of the ring. Press it gently and carefully.
4. With a quick movement turn the slide so that the cover slip is uppermost.
5. Now focus the edge of the drop under low power objective. Lower the condenser and use
concave mirror.
6. After the edge of the drop is seen in the centre of the field turn to high dry power objective

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Hanging drop preparation

Hanging drop preparation

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EXERCISES

1. Study the morphology and motility of the bacteria under high power.
2. Write the diagram of the drop edge with the bacteria.

NOTE: Bacteria when suspended in fluid show motility.

1. T rue motility is movement from one place to another and in different directions.
2. Brownian movement (false movement) is only vibration caused by molecular
bombardment of the fluid.
3. Convection current: Movement in a single direction is usually due to convection current
Motility of Bacteria is usually due to the presence of flagella.

Different types of flagellar arrangements:

Monotrichous (polar) - Vibrio cholerae

Lophotrichous (tuft at one pole) - Spirillum minus

Amphitrichous (tuft at both pole) - Pseudomonas

Peritrichous (all over the body) - Salmonella and Esch. Coli

Non motile bacilli (without flagella) eg. Klebsiella, Shigella.

Certain bacteria like spirochaetes are motile without flagella.

Other Methods of Demonstration of Motile:

Macroscopic methods: 1. Use of semisolid agar medium (Agar 0.2%)

2. Craige’s tube method

Microscopic methods: 1. Dark ground microscopy

2. Phase contrast microscopy

Demonstration of: 1. Motility in semisolid agar medium

2. Stained smear showing flagella

3. Stained smear of Spirochaetes

Questions:

1. Why do you focus the edge of the drop?


2. Which are all the motile bacteria?
3. Mention the bacteria which show motility at one temperature and remain non-motile at
another temperature.
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