Microbiology Lab (7)Chemical Agents that Affect Microbial

Growth

ABDELRAHEEM BA

A chemical substance used in treatment of infectious disease.◦ Bacteriocidal agents.

Kill bacterial cells that are actively growing.◦ Bacteriostatic agents.

Inhibit cell growth. The ultimate elimination is dependent upon host’s

phagocytic activity.

Antimicrobial agent

Antiseptics. Disinfectants. Chemotherapeutic agents (Synthetic drugs). Antibiotics.

Types of antimicrobial agents

Microbicidal or Microbistatic. Harmless enough to be applied on the skin

and mucous membranes. Should not be taken internally. Examples:

◦ Mercurials.◦ Silver nitrate.◦ Iodine solution.◦ Alcohols.

Antiseptics

Agents that kill microorganisms, but not necessarily their spores.

Not safe for application to living tissues. Used on inanimate objects (tables, floors) Examples:

◦ Chlorine.◦ Hypochlorites.◦ Copper sulfate.◦ Quaternary ammonium compounds.

Lethal for many MOs except endospores, Mycobacterium tuberculosis and non-enveloped viruses.

Disinfectants

Useful for the treatment of microbial or viral diseases.

Microbiologist’s definition of a chemotherapeutic agent requires that the agent be used for antimicrobial purposes.◦ Excluding synthetic drugs that are used to treat

diseases that are not from microbial origin. Examples:

◦ Sulfonamides (Bacteriostatic).◦ Isoniazid (treatment and prevention of TB)◦ AZT (Inhibits reverse transcriptase).◦ Chloramphenicol (broad-spectrum bacteriostatic).

Chemotherapeutic agents

Produced by MOs and are used to kill or inhibit other MOs.

Example:◦ Penicillin.

Antibiotics

Sterilization.◦ Killing all forms of life, including destruction of

spores. Disinfectants and antiseptics.

◦ Distinguished on the basis of whether they are safe for application to mucous membranes.

◦ Safety depends on the concentration of the compound. Chlorine, as added to water is safe to drink. Chlorox, an excellent disinfectant, is not safe to

drink.

Terms to know

Objectives:◦ To determine the susceptibility of bacterial growth

to selected antibiotics.◦ Helps in choosing antibiotics to treat diseases.

Antimicrobial sensitivity testing

Aka Kirby Bauer method or standardized disk susceptibility test.

Steps:◦ Inoculate bacteria on the agar surface.◦ Filter paper discs, containing known concentration

of antimicrobial agents, are placed directly on the inoculated agar.

◦ Incubation.◦ Plates are then observed for any growth

inhibition.◦ Results are reported as “Resistant (R)” or

“Sensitive (S)”.

Disk diffusion method

Disk diffusion method

Directly proportional to:◦ Amount of antimicrobial agent.◦ Solubility of the agent.◦ Diffusion coefficient.◦ Overall effectiveness of the agent.

Zone of inhibition

Mueller Hinton agar media (Thickness = 4mm).

Standard pure bacterial inoculum. Antibiotic disks. Sterile cotton swabs. Forceps.

Materials used in this test

A microbiological growth medium that is commonly used for antibiotic susceptibility testing.

Reasons for its use:◦ A non-selective, non-differential medium.

almost all organisms plated on here will grow.

◦ Contains starch. Starch is known to absorb toxins released from bacteria,

so that they cannot interfere with the antibiotics.

◦ A loose agar. This allows for better diffusion of the antibiotics than

most other plates

Mueller Hinton agar

Mueller Hinton agar

Concentration should be (1 - 1.5)*108 cell/ml.

This is equivalent to 0.5 McFarland. We have to prepare a bacterial suspension

that has a turbidity equal to 0.5 McFarland.

How to do so? Next slide.

Standard bacterial inoculum

Principle:◦ BaCl2 + H2SO4 BaSO4 (Turbid)

How to prepare 0.5 McFarland?◦ Mix 5 ml from tube #1 with 5 ml normal saline (two fold

dilution)◦ The resultant tube would have a turbidity equivalent to (1 -

1.5)*108 .

Standard turbidity

1% H2SO4

1% BaCl2

Equivalent number of bacteria

McFarland

9.9 ml 0.1 ml 3*108 cell/ml 1

9.8 ml 0.2 ml 6*108 cell/ml 2

9.7 ml 0.3 ml 9*108 cell/ml 3

9.6 ml 0.4 ml 12*108 cell/ml 4

After preparing the standard growth, soak a sterile cotton swab in the inoculum solution.◦ Remove excess inoculum by squeezing the swab on the inner

side of the tube. Move the swab all over Mueller Hinton agar in three

directions.◦ Horizontal, vertical and around the edge.

Leave the plate for 5 minutes to dry. Apply antibiotic disks over the plate using sterile

forceps.◦ Distance between disks should be at least 2.5 cm.◦ Distance between disks and edge should be at least 1.5 cm.

Incubation.

Procedure

After incubation measure the zone of inhibition (in millimeters).

There is a reference table which demonstrates the zone of inhibition for each drug in order to determine R Vs S.

Example:◦ For drug X.

“S” if the zone of inhibition >15 mm. “R” if the zone of inhibition < 10 mm. “I” if the zone of inhibition is 10-15 mm.

Procedure

Type of MO. Type of media.

◦ Mueller Hinton must be used. Agar depth.

◦ Must be 4mm. Type of antibiotic. Concentration of antibiotic. Number of bacteria.

◦ Must be (1 - 1.5)*108. pH of culture media.

◦ Mueller Hinton pH is adjusted to neutral.

Factors affecting zone of inhibition

Can be used to determine MIC & MBC.◦ Minimal inhibition concentration (MIC).

The lowest concentration of antimicrobial agent required to inhibit the growth of a bacterial isolate.

◦ Minimal bactericidal concentration (MBC). The lowest concentration of antimicrobial agent

required to kill the growth of a bacterial isolate.

Dilution Method

Make a serial dilution of an antibiotic in tubes (range: 0.1-128 mg/ml).◦ Label each tube with the concentration of

antibiotic. Add to all tubes an equal amount of a

standard inoculum. Incubate tubes for 18-24 hours. Check growth by turbidity.

◦ The concentration of antibiotic in the first tube which shows no growth after incubation is the MIC.

Procedure

MIC

640.5 1 2 4 8 16 320.25MIC

To determine MBC◦ Subculture all tubes showing no growth on a fresh

agar plate media lacking antibiotic.◦ The lowest antibiotic concentration tube from

which the MO does not recover and grow when transferred to fresh medium is the MBC

MBC

MBC