CHEMICALS EFFECTS ON BACTERIAL

GROWTH

7th Lab
WHAT FACTORS LIMIT MICROBIAL
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)
Terminology
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)
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
CHEMICALS
Chemicals 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.
Chemical Methods
Halogens (Chlorine, Iodine, Bromine)

 Extremely effective, water disinfection,

wound treatment (I2 tablets water
treatment)

 widely used antiseptics and

disinfectants; iodine acts
denatures proteins by breaking
disulfide bonds

http://www.sleever.com/article/betadine-acquires-a-
european-dimension
 ANTIMICROBIAL AGENTS

Substances produced by some microorganism

that inhibit or kill the growth of other
microorganisms

The discovery of Penicillin

Louis Pasteur: Chance favors the prepared mind
 The story of penicillin
P. chrysogenum (P. notatum)
Sir Alexander Fleming

The Nobel Prize in Physiology or Medicine 1945

"for the discovery of penicillin and its curative effect in various infectious diseases"
Antimicrobial Agents
• 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
www.nature.com
THINGS TO DO
Bauer-Kirby method of antimicrobial
susceptibility.

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.
 McFarland Standard
McFarland
0.5 1 2 3 4
Standard No.

1.0% Barium
0.05 0.1 0.2 0.3 0.4
chloride (ml)

1.0%
Sulfuric acid 9.95 9.9 9.8 9.7 9.6
(ml)
Approx.
cell
density (1- 1.5 3.0 6.0 9.0 12.0
2X10^8
CFU/mL)
%
Transmittanc 74.3 55.6 35.6 26.4 21.5
e*
Absorbance
0.08 to 0.1 0.257 0.451 0.582 0.669
*
 Antimicrobial Susceptibility Tests

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.
 TABLE 2: CLSI/VAST APPROVED INTERPRETIVE CRITERIA
FOR ANTIMICROBIALS USED IN FOOD ANIMALS ….

Zone Diameter
Concentrations (μg/ml)
(mm)
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 Iodine
CuSO3 Dettol

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
 Minimal Inhibitory Concentration (MIC)
vs.
Minimal Bactericidal Concentration (MBC)
32 ug/ml 16 ug/ml 8 ug/ml 4 ug/ml 2 ug/ml 1 ug/ml

MIC = 8 ug/ml
Sub-culture to agar medium
MBC = 16 ug/ml

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