7th lab chemicalneffect on micribialngrowth

1. 7th Lab
CHEMICALS EFFECTS ON BACTERIAL
GROWTH

2. • 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

3. 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

4. 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

5. 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

6. • 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

7. Factors that influence effectiveness
 Number of microbes
 Conc.and kind of agents used
 Time exposure
 Microbial characteristics

8. CHEMICALS
Chemicals can be used
to disinfect and sterilize
Called germicidal
chemicals
React with vital cell
structures and components
Proteins
DNA
Cell membrane

9. 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

10. 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.

11. 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

12. ANTIMICROBIAL AGENTS
The discovery of Penicillin
Louis Pasteur: Chance favors the prepared mind
Substances produced by some microorganism
that inhibit or kill the growth of other
microorganisms

13. 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

14. • 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

15. ANTIMICROBIAL AGENTS
www.nature.com

16. THINGS TO DO

17. 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.

18. 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

20. 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

21. 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

22. 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. 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