MCB2010C Lab Quiz #3

Procedure for pouring an agar plate

obtain a tube of liquefied trypticase soy agar from 60 Celsius hot water bath. Then allow the media to for a minute and then open and flame the mouth of the tube. Remove the lid from the petri dish and pour the trypticase soy agar into the the petri dish. Rotate the plate so the mixture become evenly distributed. lead the lid slightly cracked to allowed the trypticase soy agar to cool down and harden.

Why is the lid of the plate vented after pouring?

prevent condensation of the surface of the plate

Why are plates incubated upside down?

prevent water condensation from gathering on the lid and dripping onto the agar surface

Goal of quadrant streak plate method

to isolate microbes in pure culture

Steps in quadrant streak plate method

1. Label agar plate (initials/ table #, mediate, date, organism, and incubation temp

2. Divide plate into 4 quadrants

3. After flaming the inoculating loop and mouth of bacteria tube aspectically transfer the bacteria onto the 1st quadrant of the streak plate. edge to edge

4. flame the loop and allow it to cool then make 5-6 streaks from quadrant 1 to quadrant 2

5. flame loop then streak from quadrant 2 to 3

6. flame loop and then streak from quadrant 3 to 4

7. flame the entire loop

8. incubate the plate in a inverted position at 37 C for 24-48 hours.
   
   

Theory of quadrant streak plate method

diluting the mix culture across 4 quadrants of your plate. the 4th quadrant will have isolated colonies.

Be able to recognize the difference between a good streak plate and a bad

streak plate

good streak: you can identify good single colonies
Bad streak: isolated colonies are not present

What is sub-culturing? Describe how it was done.

sub culturing is when an isolated colony is picked off and transferred to a sterile medium (usually a TSA slant)

Be able to look at a gram stain and know if the sampled culture is pure or not

You will know if the sample is pure cultured if all the bacterial cells under the microscope share the exact same color, shape and arrangement. (E.coli would be bacillus shaped and pink while S. aureus would be coccus shaped and purple

Differentiate organisms present in the mixed culture presented in the PowerPoint for pure culturing and how were they differentiated?

a. Escherichia coli (cream/white color colony)

b. Staphylococcus aureus (yellow color colony)

In the petri dish staphylococcus aureus will be yellow and Escherichia coli would be cream and bigger.

lab 12

lab 12

Differentiate between direct and indirect methods for bacterial enumeration.

Direct methods count individual cells (numerical count) while indirect methods estimate cell numbers without counting (measuring a growth effect like tubidity)

Quantitative Plating Method (Standard Plate Count)

used in direct method of enumeration. SPC (standard plate count) only counts living organisms making it a viable count (living cells)

calculate dilutions and dilutions factors

serial dilutions is the process of repeatedly diluting a solution by a constant factor to reach a lower usable concentration.
Dilution factor is determined by dividing the volume factor by the total ml.

Choose correct plate to count – Calculate original cell density (# CFUs/ml)

only plates with 30-300 colonies are statistically reliable.
to determine cfu/ml original culture use this formula:
Colony count on agar plate/ vol plated (ml) x Dilution factor for tube

What does cfu stand for?

colony forming units

Relationship between absorbance, cell density, optical density and transmittance.

transmittance (%T) is the precent of how much light is passing through the sample (measured by spectrophotometers)
Optical density is the turbidity in a sample. The turbidity shows an increase in cells. When optical density increases transmittance decreases and vise versa.
Absorbance is how much light is absorbed by the molecules inside the sample
cell density is the concentration of cells suspended in the liquid.

lab 13

lab 13

Review media classification flowchart

Understand the purpose of each type of media

Trypticase soy agar (TSA): general purpose media (most microbes can grow on it)
Trypticase soy agar (TSA) + 10% NaCl: Selective media (only organisms that can grown on 5% NaCl will grow… halotolerant) prevents growth of some organism, not all. Halotolerant means it can grow in high slat concentrations but it does not need salt to live.
Blood agar: Enriched and Differential

• Enriched: for microbes that require complex organic molecules such as blood or special growth items like vitamins. fastidious- will only grow if specific nutrients are present in its culture medium.

• Differential: visually distinguish between different types of bacteria based on biochemical differences.

Know the names and classification of each medium used

Trypticase soy agar (TSA): general puurpose media
trypticase soy agar TSA + 10% NaCl (salt agar): selective media
Blood agar: Enrichment and Differential
Mannitol salt agar (MSA): selective and differential
Maconkey Agar (MAC): selective and differential

Know the selective and differential agents and pH indicators in each media

Trypticase soy agar TSA +10% Nacl (Salt Agar): is selective media. 10% na ck tolerated by halotolerant bacteria

Blood Agar: differential due to differences in blood hemolysis (breakdown)

• complete breakdown: beta hemolysis (clear zone around colony)

• Partial breakdown: alpha hemolysis (brown zone surrounding colony)

• No breakdown: gamma hemolysis (no zone around colony)

Mannitol Salt Agar MSA: selective for halotolerant organism (7.5% NaCl). Differential: mannitol (sugar) fermentation. Phenol Red pH indicator
MacConkey agar (MAC): selective for bile tolerant organism. Differential because of lactose sugar fermentation.

Know what each color reaction tells you for each media.

Mannitol salt agar (MSA): fermenters of mannitol acid lowers pH which changes the phenol red to yellow. If it is non mannitol fermenters then media stays red. Growth if halotolerant.
MacConkey agar (MAC): fermenters of lactose acid lowers pH levels which changes the natural red color to a red/pink
if it is non lactose fermenters than colonies will not turn red/pink. Growth if bile tolerant. no growth if not bile tolerant.

Know hemolytic patterns: beta, alpha and gamma

beta: complete breakdown (clear zone)
alpha: partial breakdown (brown zone)
gamma: no breakdown (no zone)

lab 14

lab 14

List 2 ways that motility can be determined in the lab.

Wet mount or by the Tube method

Know the procedure for and advantages and disadvantages of wet mount

In wet mount a drop of viable cells (living) is placed between a slide and a cover slip and then observed in darkfield microscopy. Advantage is that its super quick to do and you are able to see bacteria move in real time. A disadvantage is that wet mounts tend to dry out quickly so microscopic observations are not recommended for long periods of time.

Be able to determine “true” motility in a wet mount - Be able to distinguish between Brownian movement and motion due to flow of liquids from true motility.

true motility is when you can see directional movement of the bacterium. Bacteria will also be moving in different directions. Brownian movement is caused by invisible molecules bombarding bacterial cells. If the movement you see is vibrational and not directional then the organism is non motile.

Name and describe the media used in the tube method of motility determination.

a. Know the agar concentration of semi-soft agar (Motility Media)

b. Know the special ingredient in the Motility Media that creates a red

precipitate and the reason the composition of the red precipitate forms.

a. the semi soft agar is called motility test medium/ motility media. lower content of agar

b. triphenyl tetrazolium chloride (TTC) is the special ingredient in motility media that created a red precipitate. the reduced salt of TTC is red (occurs as a result of bacterial metabolism and indicated where bacterial growth has occured).

Know how was this media inoculated.

inoculating needle

Know names (genus and species) of motile and non-motile organisms we used in lab

Staphylococcus aureus was non motile while Proteus Mirabilis is motile

lab 15

lab 15

Know the 5 main classifications of oxygen requirements in microorganisms.

Obligate aerobes: requires o2 for growth

obligate anaerobes: can not survive in oxygen. use fermentation or anaerobic respiration

Facultative anaerobes: can grow in in oxygen or without (us ) use oxidative respiration and fermentation

Microaerophiles: require small amounts of oxygen (5-15%).

Aerotolerant anaerobes can survive in o2 due to presence of superoxide dismutase but do not use o2 as a terminal electron acceptor.

Full name of the medium we used to determine oxygen requirements

Fluid Thioglycolate Medium (FTM)

Know component of the medium that is a reducing agent (removes oxygen)

sodium thioglycolate Medium removed oxygen

Know why is medium boiled before inoculation?

to drive out dissolved oxygen and create a anaerobic reaction.

What is the name of the indicator dye that detects oxygen in the media?

Resazurin (pink in presence of ox)

Be able to observe growth patterns to evaluate oxygen requirements

obligate aerobe: organism growth at the top of the medium

facultative anaerobe: organism growth throughout the medium

obligate anaerobe": organism growth at the bottom of the medium

Know the names (genus and species) of the organisms we used in lab to

demonstrate each type of oxygen requirement demonstrated.

Clostridium sporogeneses : anaerobe
Escherichia coli : facultative
Pseudomonas aeruginosa : aerobic

What are the 2 enzymes lacking in an obligate anaerobe?

superoxide dismutase and catalase