MONTGOMERY COUNTY COMMUNITY COLLEGE
Department of Science
BIO 140
CHAPTER 6
MICROBIAL GROWTH
I.
MEDIA (PLURAL) OR MEDIUM (SINGULAR)
A.
B.
Also Refer to Lab Manual
Lab 8 and Appendix A
Growing Chemoheterotrophs
1.
General nutrients
2.
General guidelines
a.
carbon source
b.
energy source
c.
other nutritional needs
Solid Vs. Liquid
1.
Advantages of liquid
a.
b.
c.
2.
Solid = addition of agar (a complex _______________ obtained from
_______________.)
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3.
Advantages of solid
a.
b.
c.
4.
Characteristics of agar
a.
b.
c.
d.
e.
C.
Nutritive
1.
Definition: Medium that in general will provide nutrients required to grow
most _______________ and _______________.
Basic growth nutrients supplied as peptones and meat or yeast extracts.
2.
Examples
a.
Trypticase Soy Agar (TSA) Ingredients
1)
2)
3)
4)
5)
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b.
D.
Trypticase Soy Broth (TSB)
Enriched
1.
Definition: Contains general supportive medium to which nutrients
__________________________________________________________
2.
Examples
a.
Blood Agar (BAP)
Composed of TSA plus 5% SB
b.
Chocolate Agar (CAP)
BAP plus additional growth factors
Heating the sheep blood releases extra nutrients and gives the
plate its “chocolate” appearance
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E.
Selective
1.
Definition
2.
Examples
a.
b.
c.
Colistin Naldixic Acid Agar (CNA)
1)
Colistin
2)
Naldixic Acid
MacConkey’s Agar (MAC)
1)
Bile Salts
2)
Crystal Violet (Dye)
Mannitol Salt Agar (MSA) – Refer to Lab Manual
Salt – 7.5% NaCl
F.
Differential
1.
Definition: Visible difference in colonies.
2.
Examples
a.
Blood Agar – shows hemolysis (lysis of red blood cells)
1.
Alpha hemolysis
2.
Beta hemolysis
3.
Non-hemolytic
b.
MacConkey’s
Lactose
c.
Mannitol Salt Agar
Mannitol
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G.
Media That Are More Than One Category
1.
Enriched and Differential = _____________________________________
2.
Selective and Differential = ____________________________________
____________________________________
II.
PHYSICAL REQUIREMENTS
A.
Temperature
1.
Psychrophiles/Psychotrophs
Temperature range: __________________________________________
Optimum temperature: ________________________________________
2.
Mesophiles
Temperature range: __________________________________________
Optimum temperature: ________________________________________
3.
Thermophiles/Extreme Thermophiles
Temperature
Range:
T: ______________________________________________
ET: _____________________________________________
Optimum:
T: ______________________________________________
ET: _____________________________________________
B.
pH
1.
Ideal bacterial pH ____________________________________________
2.
Ideal pH for fungi ____________________________________________
3.
Neutralizing acids
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C.
Osmotic Pressure – see Text
1.
Isotonic – the concentration of solutes inside and outside of the cell are
equal
Salt (NaCl) concentration = ____________________________________
2.
Hypertonic
Salt Concentration = _________________________________________
3.
Hypotonic
Salt Concentration = _________________________________________
4.
D.
Define plasmolysis:
Chemical Requirements: Classification based on oxygen requirements
1.
Aerobes – Microbes that use molecular oxygen
2.
Anaerobes
a.
b.
c.
microbes that are unable to use molecular oxygen
most are harmed by O2
grown in anaerobe jar
3.
Facultative Anaerobes
4.
Microaerophiles/Capnophiles
5.
a.
definition
b.
requires decreased O2/increased CO2
c.
grown in _____________________________________________
d.
or grow in ____________________________________________
Define the term obligate
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III.
CULTURE TECHNIQUES
A.
Streak Plate Technique (for most bacteria)
1.
2.
3.
4.
5.
6.
7.
B.
Other Culture Techniques
1.
Animals
2.
Living cells
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IV.
BACTERIAL GROWTH
A.
B.
Binary Fission – Usual Method
1.
Cell preparation
2.
Generation time
Bacterial Growth Curve (In a closed system)
1.
The Lag Phase
2.
The Log Phase
3.
The Stationary Phase
4.
The Death Phase
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V.
COUNTING MICROORGANISMS
A.
Direct Measurement
1.
B.
Plate Count
a.
Spread Plate
b.
Pour Plate
2.
Filtration
3.
Microscope Count using a special “slide”
Indirect
1.
A Spectrophotometer – Turbidity
2.
Metabolic activity
3.
Measure dry weight (for filamentous fungi)
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VI.
SPECIMEN COLLECTION (Refer to Appendix B – Textbook)
A.
Site – Disinfection
B.
Container
C.
1.
Liquid, body fluids
2.
Aspirate
3.
Tissue
4.
Swab
5.
Hair, nail clippings
Transport
1.
Moisture
2.
pH
3.
Temperature
4.
Gaseous environment
5.
Osmotic pressure
6.
Time till arrival in laboratory
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D.
E.
Timing Of Specimen Collection
1.
Prior to antibiotics
2.
When the organism is present
3.
When patient spikes a temperature
4.
When in doubt ? ? ? ?
Other Criteria For An ACCEPTABLE SPECIMEN
Revised 2/2009
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CHAPTER 7
CONTROL OF MICROBIAL GROWTH
I.
HISTORY
A.
B.
1840-1860
1.
Contribution of Ignatz Semmelweis
2.
Contributions of Joseph Lister
1860-1898
1.
2.
3.
4.
C.
Changes Since 1898
1.
2.
3.
4.
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II.
III.
DEFINITIONS
A.
Sterilization
B.
Disinfection: Removal of vegetative pathogens; more resistant forms may not be
killed (endospores and cysts).
C.
Antiseptic: A disinfectant that may be used on living tissue.
D.
Sepsis
E.
Static: Stops growth: once the agent is removed, growth might resume.
F.
Cidal
CONSIDERATIONS WHEN SELECTING A METHOD OF STERILIZATION
A.
Material To Be Sterilized
B.
Cost/Availability
C.
Number Of Organisms/Microbial Load
D.
Nature Of The Organisms
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E.
DECREASING ORDER OF RESISTANCE OF MICROORGANISMS TO
CHEMICAL ANTIMICROBIALS
ORGANISM
UNIQUE CHARACTERISTIC(S)
1.
Prion
Size
2.
Bacterial Endospores
Extremely Resistant Endospore Coat
3.
Bacteria – Mycobacterium
Tuberculosis
Fatty Acids In Cell Wall
4.
Cysts Of Protozoa
5.
Vegetative Protozoa
6.
Gram Negative Bacteria
7.
Fungi
Fungal Spores
8.
Viruses
Non-Enveloped
9.
Gram Positive Bacteria
10.
Viruses - Enveloped
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IV.
METHODS OF STERILIZATION
Method
Heat
1. Autoclave
Description
Temperature
Moist Heat –
Steam
Method of
Action
Preferred Use
Denatures
Protein
2. Incinerator
3. Hot Oven
4. Cold Chemical
Glutaraldehyde
(Cidex)
Denatures
Protein
5. Gaseous
Sterilizers
Inactivates
Protein
Ethylene Oxide
6. Other Gases
7. Filtration
HEPA
8. Membrane
9. Gamma
Radiation
10. Ultrasound
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V.
METHODS OF DISINFECTION
A.
Conditions Influencing Microbial Control
1.
2.
3.
4.
5.
6.
B.
Types Of Disinfectants
Chemical Agent
1. Soap & Water
Reg. Soap
Mechanism of
Action
Antibacterial
2. Alcohol
3. Halogens
Iodine
Denatures
Protein
Dissolves
Cell Membrane
Denatures
Protein
Oxidizes
Tincture of
Iodine
Chlorine Gas
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Preferred Use
Comments
Chemical Agent
4. Phenolics
Mechanism of
Action
Denatures
Proteins
Preferred Use
Comments
Disrupts
Cell Membrane
5. Chlorhexadine
6. Quaternary
Ammonium
Compounds
“Quats”
Probably Affect Cell
Membrane
7. Aldehydes
Denatures Protein
8. Heavy Metals
Copper
Silver Nitrate
Mercury
Inactivates Protein
9. Hydrogen
Peroxide
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Example: Zephiran
(Benzalkonium
Chloride)
VI.
PHYSICAL CONTROL
Method
A. Boiling
Mechanism of
Action
Moist Heat Denatures
Protein
Preferred Use
Comments
Temp ° C: _________
Temp ° F: _________
B. Pasteurization
1. Classic
Time: _____________
Temp: ____________
2. HTST
Time: _____________
Temp: ____________
3. UHT
Time: _____________
Temp: ____________
C. Refrigeration
4-10° C
Stops Enzyme Activity
D. Freezing
Below 0° C
Preserves Organisms
E. Dessication
“Dries Out”
Disrupts Metabolism
F. Osmotic Pressure
1. Bacteria
Plasmolysis
2. Yeasts And
Molds
G. Sorbic And Benzoic
Acid
Revised 2/2009
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