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Glossary of Micro Lab - 3053

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Created by daisy497

Medium
mixture of nutrients
Agar
non-nutritive solidifying agent
Culture
bacteria that grow and multiply in a container of medium
Incubation
Used to stimulate growth of bacteria
- pathogens (37 degrees)
- environmental microbes (~25 degrees)

Incubation time depends on generation time:
- generation time 10-15 mins (12-24 hours)
- generation time several hours (72 hours or longer)





Generation time
one complete cell division
How to achieve aseptic technique?
1. Keep containers closed
2. Sterilize loop with flame until red hot before and after use
3. Pass the mouth of tube over flame after opening and before closing
4. Clean desk with Endbac before and after use
5. Do not place tube cap on table while inoculating. Hold under little finger.
6. Hold tube as horizontal as possible when inoculating
7.
6.






How to describe agar slants?
- Amount of growth: no growth, slight, scant, moderate, very good/profuse
- color
- opacity (opaque, translucent, transparent)

How to observe liquid broth?
- turbid, cloudy, clear
- pellicle (at the surface) or sediment (at the bottom)
****What is MAGNIFICATION?
- depends on the type of objective lens used with the ocular lens
- TOTAL MAGNIFICATION = objective lens magnification x ocular lens (ex. 40X x 10X)
*****What is RESOLUTION (Resolving Power)?
- the ability of the lenses distinguish 2 points that are very close together as distinct and separate
What does Resolution (resolving power) depend on?
1. Numerical aperature
2. Wavelength
3. Design of Condenser

What are the course and fine focuses?
moves the stage up and down to bring the specimen into focus
*What is the condenser?
concentrates the light before it passes through the specimen
What is the iris diaphragm?
regulates the amount of light that enters the condenser lens
Ocular lens
- lens that passes the image to the eye
- magnifies image 10x
What is a Rheostat?
adjusts the intensity of the light
What is the purpose of Oil Immersion?
- increases the resolution (resolving power)
- prevents light refraction

*when using, OPEN the iris diaphragm as much as possible
*condenser should be at HIGHEST position



Morphology
- shape (cocci, bacilli, spirals)
Characteristics of a good smear?
1. Not too thick or too thin:
- if too thick, will not be able to clearly identify size, shape and arrangement & staining procedure may be affected
- if too thin, may be difficult to focus the slide. It may also be difficult to identify the arrangement of cells
2. Allow slide to air dry completely before heat fixing - to prevent distortion of cells
3. Ensure proper heat fixing - to ensure it can endure repeated washing



What is the purpose of heat fixing?
1. to kill the bacteria
2. to cause the bacteria to stick to the glass slide
Gram Stain procedure:
1. Primary Stain: Crystal Violet (1 min & rinse) - all PURPLE
2. Mordant: Iodine (1 min & rinse) forms CV-I complex - all PURPLE
3. Decolorizing agent: 95% Ethanol (10 secs & rinse; repeat - 20 secs total) - gram pos: PURPLE; gram neg: colorless
4. Counterstain: Safranin (30 Seconds, rinse and blot dry) - gram pos: PURPLE; gram neg: PINK


What is the most important step in gram staining?
Decolorization
Why is it important that crystal violet precede the iodine mordant?
Iodine by itself has little affinity for bacterial cells
What happens if decolourizer is left on the smear too long?
- some gram positive cells may be decolourized and appear gram negative
What is a simple stain?
- a single stain (such as methylene blue or crystal violet) is used to dye the bacteria
- size, shape and arrangement can be determined but all organisms are stained the same colour
What is a differential stain?
- 2 contrasting stains used to visualize special structures of bacteria (such as flagella or endospores)
- to separate bacteria into groups (ex. Gram staining and acid-fast)
Characteristics of Gram Positive cells?
1. Contain large amounts of peptidoglycan (50-80%)
2. Peptidoglycan provides barrier in which CV-I complex cannot pass during decolorization
3. CV-I complex is retained and cells appear purple after counterstaining

Characteristics of gram negative cells?
1. Contain little peptidoglycan but appreciable amounts of lipid (10-30%)
2. Alcohol is a solvent of lipids and extracts the lipids from the cell wall
3. Cell wall becomes porous and permeable, allowing for the loss the CV-I complex

Which bacteria do not possess a cell wall?
Mycoplasmas
What factors affect gram staining results?
1. Smear preparation
2. Concentration and freshness of reagents - may affect quality of stain
3. Washing and drying of the smear excess water can dilute reagents
4. cultures that are in exponential phase of growth (18-24 hours old) - older cultures contain ruptured and dead cells
5. pH of the culture medium



What is the streak plate method?
diluting the # of bacterial cells present until we get a single, isolated colony "pure culture"
What is acid-fast bacteria?
- contain waxy lipids in the cell wall (becomes impermeable to most stains)
- acid-fast staining is facilitated by heat to soften the lipids to allow stain to penetrate (once stained, they resist decolorization with acid alcohol)
What are examples of acid-fast bacteria?
MYCOBACTERIUM (M. smegmatis; M. leprae - leprosy; M. tuberculosis - TB)

What is Carbol fucshin?
- acid-fast stain
- soluble in lipids
- therefore, Carbol fuchsin is retained in the cell wall of acid-fast bacteria but washed out with acid alcohol in non-acid fast bacteria

Acid-fast staining procedure
1. Place slides in fumehood
2. Flood with Carbol fuchsin (6 MINS)
3. add more stain as evaporation occurs
4. pour off stain and rinse (in fume hood)
5. lay slides on rack near sink
6. Flood with acid alcohol (15 SECS & rinse; repeat) - to decolourize
7. Counterstain: Methylene Blue (1 MIN, rinse & blot dry)
8. Examine under oil immersion








Results of acid-fast staining?
- acid-fast: fuchsia
- non-acid fast: Blue
Endospores are important structures for the survival of which 3 genera of GRAM POSITIVE?
1. Bacillus
2. Clostridium
3. Sporosarcina

Characteristics of spores?
1. high resistance (related to dehydrated state inside spore and DPA which is a chemical that allows spores to survive at high temps and with harsh chemicals)
2. Spores can be dormant for hundreds of years
3. Do not reproduce (survival only) - one endospore/cell
4. Length of time for spores to form varies from species to species
5. Size: greater than, less than or equal to diameter of cell
6. Shape (spherical, oval, or cylindrical)
7. Position (central, terminal or subterminal)





What is the "spore coat"?
- part of an endospore
- relatively impermeable proteinaceous (surrounds cortex)
Endospore staining procedure
1. Place slides on rack
2. flood with MALACHITE GREEN (5 MINS)
3. Heat slide underneath with flame
4. add more reagent when evaporation occurs
5. Rinse slide (water is decolourizer for vegetative cells)
6. Counterstain: Safranin O (1 MIN, Rinse & blot dry)
7. Examine under oil immersion





Results of endospore staining?
- Endospores: green
- Vegetative cells: pink
What is a Synthetic (Defined) Media?
- exact composition is known
- duplication is possible
What is a complex media?
- exact composition is unknown
- exact duplication is NOT possible
- ex. (yeast or beef extracts)

What is an Obligate aerobe?
- grow only in the presence of free O2
What is a facultative anaerobe?
- grow in the presence or absence of free O2
What is a Obligate anaerobe?
- grow only in the absence of free O2 (O2 is lethal to these organisms)
What are Microaerophiles?
- require low O2 concentration (not common)
Which organisms produce catalase and superoxide dismutase enzymes?
- Obligate aerobes
- Facultative Anaerobes

*these enzymes allow microbes to use O2 and continue growing without accumulating toxic forms of oxides that would kill them


What does a reagent sachet contain?
1. Ascorbic acid - reduces O2 level
2. Activated Carbon
3. Inorganic Carbonate - heat causes inorganic carbonate to decompose and release sufficient CO2
4. Water


When are minimum and maximum temps measured?
- 72 hours
What are optimum temps measured?
24 hours
3 primary groups of microbes based on growth at various temperatures?
1. Psychrophiles (0-15 degrees)
2. Mesophiles (25-40 degrees)
3. Thermophiles (45-65 degrees)

What are FASTIDIOUS organisms?
- require only 1 or 2 vitamins or amino acids
What is Selective Media?
- permit some bacteria to grow but not others
- contain 1 or more components that suppress growth of some microbes (ex. dyes, salts, unfavourable pH, antibiotics, etc.) without affecting the ability of others to grow
What is Differential Media?
- contain 1 or more components (such as a particular carbohydrate) that can be used by some microbes but not others
- a change will occur in the medium (ex. colour change or rxn)
What is Enrichment Media?
- contain all the nutrients required for growth
- increase bacterial #'s and grow fastidious orgnaisms

- ex. Chocolate agar & Blood agar
- plasma & RBCs provide excellent enrichment



What is an undefined (complex or rich) medium?
- the exact amounts and and kinds of large organic molecules are unknown
- support the growth of bacteria better because they contain more preformed nutrients
What is a defined medium?
- made up of specific amounts of chemicals
- the exact components are known
What are 3 primary purposes of a battery?
1. to determine the which bacteria PREDOMINATES
2. RESPONSES to various ingredients of culture media
3. to SELECTIVELY encourage growth of those species while suppressing normal flora

What are 3 possible results when observing Blood Agar Medium?
1. Alpha: incomplete hemolysis (greenish color of media in area of growth)
2. Beta: complete hemolysis (clean area surrounding growth)
3. Gamma: no change in bacteria or medium (absence of toxins)

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