lab 3 Flashcards
What is the name of the microscope that will be used to observe close up details of agar colonies?
Light microscope
Darkfield microscope
Dissecting microscope
Phase Contrast Microscope
Dissecting microscope
When inoculating the biochemical test tubes, you will use
Both inoculating loop and needle
Inoculating loop
Inoculating needle
Pasteur pipette
Both inoculating loop and needle
When performing the streak plate, it is important to sterilize the loop each time between quadrants.
TRUE OR FALSE
TRUE
Following incubation the streak plate has no growth in quadrants 2, 3, and 4. The primary quadrant 1 has extensive growth throughout.
Choose a possible reason for this below.
1 The bacteria’s optimal incubation temperature was not achieved.
2 Inoculation loop was too hot and killed the bacteria when “streaking” out quadrant two (B).
3 Inoculation loop was contaminated with bacteria from the air.
4 The inoculation loop was too cool and the bacteria will not divide.
Inoculation loop was too hot and killed the bacteria when “streaking” out quadrant two (B).
The mutations experiment being conducted during Lab 3 includes:
1 control plate and 4 treatment plates
3 treatment plates
5 treatment plates
2 duplicate control plates and 3 treatment plates
1 control plate and 4 treatment plates
When describing colonies growing on agar, how many characteristics are we using to describe them?
One Two Three Four Five
Five
Which of the following is an example of a negative (anionic) stain that is repelled by bacterial cytoplasm?
Select Answer:
Nigrosin
Safranin
Crystal violet
Methylene blue
Nigrosin
Extensive growth was observed in all 4 quadrants of a streak plate, but no isolated colonies were observed at all in any of the quadrants following incubation. Choose a possible reason for this below. (note: isolated colonies are those that are separated from other colonies and a good steak plate will typically have isolated colonies in quadrants 3 and 4).
1 Flaming of the inoculation loop did not occur between quadrants.
2 The inoculating loop was not held at a slight angle.
3 The inoculating loop did not enter the previous quadrant before streaking out the current one.
4 The inoculation loop was sterilized prior to streaking each quadrant.
Flaming of the inoculation loop did not occur between quadrants.
MacConkey agar composition:
- neutral red (pH indicator dye): neutral pH - medium is a light pink color; acidic pH - dark pink medium color; alkaline pH - medium is beige)
- crystal violet (inhibits Gram positive bacteria)
- bile salts (inhibits Gram positive bacteria and fastidious (Gram negative bacteria)
- lactose (fermentable disaccharide providing carbon and energy; bacteria fermenting the sugar produce acids which lower the pH of the medium)
- 0.5% sodium chloride (electrolyte transport and osmotic balance)
- agar (solidifying agent)
- pH of medium after preparation is ~7.0 (medium will be a light pink color)
Given the above composition of MacConkey agar, this medium is considered ______________
(classification of medium).
- selective and differential
- Differential
- Enriched
- Selective
- General
selective and differential
In lab 1, you prepared microscope slides using a technique called wet mount preparation. Instead of wet mounts, sometimes a sample is air dried and then heat fixed in preparation for certain staining techniques. Air drying is typically done by placing the slide on the slide holder of the bacticinerator. Heat fixing is done by by placing the slide in front of the opening of the bacticinerator for 5-7 seconds.
Heat fixing bacteria on a slide leads to-
Select Answer:
1 adherence of the organisms to the slide.
2 death of the organisms.
3 altering of the organisms to more readily retain the dyes.
4 All of the choices
5 None of the choices
All of the choices
MacConkey agar composition
- neutral red (pH indicator dye): neutral pH - medium is a light pink color; acidic pH - dark pink medium color; alkaline pH - medium is beige)
- crystal violet (inhibits Gram positive bacteria)
- bile salts (inhibits Gram positive bacteria and fastidious (Gram negative bacteria)
- lactose (fermentable disaccharide providing carbon and energy; bacteria fermenting the sugar produce acids which lower the pH of the medium)
- 0.5% sodium chloride (electrolyte transport and osmotic balance)
- agar (solidifying agent)
- pH of medium after preparation is ~7.0 (medium will be a light pink color)
Bacterial Colony Characteristics
SIZE?
Estimate large, medium, small and pinpoint
Bacterial Colony Characteristics
COLOR?
Use simple terms
Bacterial Colony Characteristics WHOLE COLONY (surface)?
Circular, irregular, filamentous, rhizoid, punctiform
Bacterial Colony Characteristics
ELEVATION?
Raised, convex, umbonate, crateriform, flat
Bacterial Colony Characteristics
MARGIN (outer edge of colony, use dissecting scope)
Entire, undulate, filamentous, curled, lobate
UV exposure time? What happens?
Depending on the exposure time, the induced mutation will result in mutations that range from altered colony characteristics (color changes) to lethal mutation where death occurs
Names and classification of media
GENERAL PURPOSE?
Supports growth of wide range of non fastidious (no nutrients provided) organisms. Ex. Nutrient agar (NA)
Names and classification of media
SELECTIVE
Supports growth of specific bacteria and inhibits growth of others. Ex. MacConkey (MAC), Mannitol Salt agar (MSA), Sabouraud dextrose agar (SDA)
Names and classification of media
DIFFERENTIAL
Shows the difference between organisms grown on it. Ex. MacConkey, Mannitol Salt agar (MSA), Blood agar (BA or SBA)
Names and classification of media
ENRICHED
supports growth of fastidious organisms; different than selective in that it doesn’t usually inhibit growth of other organisms. Ex. Blood agar (same as NA but with blood)
MacConkey agar. Selective vs Differential
Selective: contains inhibitors (bile+CV) → inhibits most gram positives
Differential: contains lactose= differentiates lactos fermenters vs non fermenters.
*if fermenters present →lactose →lactic acid → pH change.
*pH indicator in medium called neutral red changes from colorless to red and below 6.8pH
Citrate Slant tube
Contains: citrate as sole carbon source and Bromothymol blue (pH indicator)
Some microbes can use citrate (aka citric acid) as sole carbon source
If citrate is used, this acid is slowly removed from the agar and the pH increases causing the pH indicator to change from green to blue
Carbohydrate (glucose or lactose) fermentation tubes
Contains less than 1% of glucose or lactose and phenol red (pH indicator)
Some microbes ferment glucose or lactose
If occurs:
Acids are produced lowering the pH; pH indicator changes from red to yellow
Gas may also be produced and some trapped in inverted Durham tube
Urea broth tube
Contains urea broth and phenol red (pH indicator)
Some microbes produce the enzyme urease.
Urease converts urea to ammonia and gas; pH increases; pH indicator dye changed from salmon to rose/dark pink
SIM agar tube “S”
Sulfide production.
Contains cysteine and iron (Fe)
Some microbes produce enzymes that hydrolyze cysteine (amino acid) into alanine and hydrogen sulfide (H2S or HS ions)
H2S or HS ions binds to iron ions (Fe) to produce iron sulfide (FeS) which causes the agar to turn black
SIM agar tube “I”
Indole production
Contains tryptophan
Some microbes produce the enzyme tryptophanase
Enzyme breaks down tryptophan to indole, pyruvic acid and ammonia
Indole is detected by adding Kovac’s reagent; if present, reagent will undergo a chemical change to a red quinoial compound.
