1. Chemical Agents that Affect Microbial Growth

2.  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.

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

4.  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.

5.  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.

6.  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).

7.  Produced by MOs and are used to kill or inhibit other MOs.  Example: ◦ Penicillin.

8.  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.

9.  Objectives: ◦ To determine the susceptibility of bacterial growth to selected antibiotics. ◦ Helps in choosing antibiotics to treat diseases.

10.  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)”.

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

13.  Mueller Hinton agar media (Thickness = 4mm).  Standard pure bacterial inoculum.  Antibiotic disks.  Sterile cotton swabs.  Forceps.

14.  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

16.  Concentration should be (1 - 1.5)*10 8 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.

17.  Principle: ◦ BaCl 2 + H 2 SO 4 BaSO 4 (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)*10 8. 1% H 2 SO 4 1% BaCl 2 Equivalent number of bacteriaMcFarland 9.9 ml0.1 ml 3*10 8 cell/ml1 9.8 ml0.2 ml 6*10 8 cell/ml2 9.7 ml0.3 ml 9*10 8 cell/ml3 9.6 ml0.4 ml 12*10 8 cell/ml4

18.  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.

19.  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.

20.  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)*10 8.  pH of culture media. ◦ Mueller Hinton pH is adjusted to neutral.

21.  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.

22.  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.

24.  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