7th Lab

CHEMICALS EFFECTS ON BACTERIAL

GROWTH

• Physical Control Methods

• Heat: Moist vs. Dry

• Autoclaving, pasteurization

• Filtration

• Cold

• Desiccation & high osmotic

pressure

• Radiation (UV, gamma rays

Chemical Control Methods

• Halogens (Chlorine,

iodine, bromine)

• Alcohols (e.g.. isopropyl

alcohol)

• Heavy metals (Ag, Hg, Cu,

Zn)

• Phenol

• Quaternary Ammonium

Cmpds (quats)

• Aldehydes (e.g..

formaline)

WHAT FACTORS LIMIT MICROBIAL

GROWTH

Bacteriostasis - Halts growth but not killed

(Ex: Refrigeration, dyes in food)

• -- cide

a suffix indicating that the agent will kill the kind of organism in question (e.g., viricide, fungicide).

• -- static

a suffix indicating that the agent will prevent the growth of the type of organism in question (e.g., bacteriostatic, fungistatic).

Asepsis - Absence of pathogens; aseptic techniques

(Ex: Air filtration, UV light, gloves)

Terminology

Sterilization

• Removal of all microorganisms

• Sterile item is absolutely free of microbes, endospores and viruses

• Can be achieved through filtration, heat, chemicals and irradiation

Disinfection

• Eliminates most pathogens

• Some viable microbes may exist

• Disinfectants - used on inanimate objects and surfaces

• Antiseptics - used on living tissues

Pasteurization

• Brief heat treatment used to reduce organisms that cause food spoilage

Decontamination

• Is the process of cleansing an object or substance to remove

contaminants such as micro-organisms

Degerming

• Mechanism to decrease the number of microbes in a specific area

• Particularly the skin

Preservation

• Process used to delay food spoilage . Often includes the addition of

growth-inhibiting ingredients

• Sanitization

• Reduction of the microbial population to a safe

level as determined by public health standards.

• ANTIBIOTICS :

• Substances produced by some microorganism

that inhibit or kill the growth of other

microorganisms

Factors that influence effectiveness

Number of microbes

Conc.and kind of agents used

Time exposure

Microbial characteristics

CHEMICALSChemicals can be used

to disinfect and sterilize

Called germicidal

chemicals

React with vital cell

structures and components

Proteins

DNA

Cell membrane

CHEMICAL METHODS -DISINFECTANTS

• Heavy Metals

• Ag, Hg, Cu

• Act by combining with proteins and inactivating them.

• (CuSO4) potent against algae in swimming pools, fish tanks.

• ZnCl2 is a common ingredient in mouthwashes

CHEMICAL METHODS

• Phenolics: laboratory and

hospital disinfectants; act by denaturing proteins

• Alcohols. Ethanol, isopropanol

• Widely used disinfectants and antiseptics; will not kill endospores; act by denaturing proteins and possibly by dissolving membrane lipids.

Halogens (Chlorine, Iodine, Bromine)

Extremely effective, water disinfection, wound treatment (I2 tablets water treatment)

http://www.sleever.com/article/betadine-acquires-a-

european-dimension

Chemical Methods

widely used antiseptics and disinfectants; iodine acts

denatures proteins by breaking disulfide bonds

ANTIMICROBIAL AGENTS

The discovery of PenicillinLouis Pasteur: Chance favors the prepared mind

Substances produced by some microorganism

that inhibit or kill the growth of other

microorganisms

The story of penicillin

P. chrysogenum (P. notatum)

The Nobel Prize in Physiology or Medicine 1945

"for the discovery of penicillin and its curative effect in various infectious diseases"

Sir Alexander Fleming

• Antibiotics are either:

• Natural, semi-synthestic or synthetic

• Natural antibiotics are synthesized by molds and bacteria

• Antibiotics made by streptomyces such as:

• Streptomycin, neomycin, tetracycline, chloramphenicol, erythromycin,

• Antibiotics produced by Bacillus sp. Such as

• Bacitracin, gramicidin, tyrocidin.

Antimicrobial Agents

ANTIMICROBIAL AGENTS www.nature.com

THINGS TO DO

1. Prepare a standard turbidity inoculum of the test bacterium so

that a certain density of bacteria will be put on the plate.

2. Inoculate a 150mm Mueller-Hinton agar plate with the

standardized inoculum so as to cover the entire agar surface with

bacteria.

3. Place standardized antibiotic or chemicals discs on the plate.

4. Incubate the plate at 37°C for 24 hours.

5. Measure the diameter of any resulting zones of inhibition in

millimetres (mm).

6. Determine if the bacterium is susceptible, moderately

susceptible, intermediate, or resistant to each antimicrobial

agent.

Bauer-Kirby method of antimicrobial

susceptibility.

McFarland

Standard No.0.5 1 2 3 4

1.0% Barium

chloride (ml)0.05 0.1 0.2 0.3 0.4

1.0%

Sulfuric acid

(ml)

9.95 9.9 9.8 9.7 9.6

Approx.

cell

density (1-

2X10^8

CFU/mL)

1.5 3.0 6.0 9.0 12.0

%

Transmittanc

e*

74.3 55.6 35.6 26.4 21.5

Absorbance

*0.08 to 0.1 0.257 0.451 0.582 0.669

McFarland Standard

Figure 43.1 A Kirby-Bauer Plate. A Mueller-Hinton agar

plate inoculated with S. aureus and various antibiotics. Notice the

diameter of the various zones of inhibition.

Antimicrobial Susceptibility Tests

TABLE 2: CLSI/VAST APPROVED INTERPRETIVE CRITERIA

FOR ANTIMICROBIALS USED IN FOOD ANIMALS….

Zone Diameter

(mm)Concentrations (μg/ml)

Antimicrobial S I R S I R

Clindamycin2 (used for lincomycin testing) ≥21 15-20 ≤14 ≤0.5 1-2 ≥4

Erythromycin3 ≥23 14-22 ≤13 ≤0.5 1-4 ≥8

Gentamicin ≥15 13-14 ≤12 ≤4 8 ≥16

Oxacillin ≥13 11-12 ≤10 ≤2 --- ≥4

Oxytetracycline ≥19 15-18 ≤14 ≤4 8 ≥16

Penicillin4 ≥28 20-27 ≤19 ≤0.12 0.25-2 ≥4

Sulfathiazole ≥17 13-16 ≤12 ≤256 --- ≥512

Tetracycline5 ≥19 15-18 ≤14 ≤4 8 ≥16

Trimethoprim/Sulphamethoxazole6 ≥16 11-15 ≤10 ≤0.5/9.5 -- ≥4/76

EFFECT OF CHEMICAL AGENTS

Water

DettolListerine

Iodine

Label the plate with the chemicals used

and bacteria species

Inoculate the plate with your bacteria

Use sterile forceps to blot the disk into

the inoculated plate

23

1 ug/ml

MIC = 8 ug/ml

MBC = 16 ug/ml

Minimal Inhibitory Concentration (MIC)

vs.

Minimal Bactericidal Concentration (MBC)

32 ug/ml 16 ug/ml 8 ug/ml 4 ug/ml 2 ug/ml

Sub-culture to agar medium