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Microbiology LAB > Lab 6 > Flashcards

Lab 6 Flashcards

1
Q

What are the various methods of achieving isolation

A

Serial dilution, streak plate method, pour plate/spread plate method, enrichment culture, selective media, and differential media

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2
Q

Describe serial dilution

A

A mixed culture could be serially diluted to the point of extinction to obtain only one single microorganism.
Useful to isolate a microorganism that is predominant in a mixed culture

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3
Q

Describe streak plate method

A

a small amount of mixed culture streaked to obtain separation on a solid medium

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4
Q

Describe pour plate/spread plate method

A

serially diluted mixed culture could be spread or poured over the surface of a solid medium

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5
Q

Describe enrichment culture

A

designed to enhance the growth of one or more types of organisms from a sample in which a number of different organisms may be present.
Useful is microbe of interest is present in very small numbers or its growth is slow relative to the other species in the sample

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6
Q

Describe selective media

A

by inhibiting the growth of other microorganisms in a sample, one can isolate organism of interest (ex. mannitol salt)

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7
Q

Describe differential media

A

by identifying it on the basis of colony characters or colors one can isolate an organism of interest. Media usually contain agents that help differentiation (ex. phenol red)

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8
Q

What are the two major steps for obtaining pure cultures from a mixed population?

A
  1. Mixture must be diluted until the various organisms become separated far enough on the agar that after incubation they form isolated visible colonies (isolation plate)
  2. Isolated colony picked off and trans. to new sterile medium. After incubation, all organisms will be descendants of the same organism (pure culture)
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9
Q

How do you determine the number of bacteria per ml after conducting pour plate counts?

A

(colonies counted)/(dilution of tube x amt plated)
OR
(colonies counted)/(plate dilution)

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10
Q

What is the purpose of the streak plate method

A

To isolate bacteria, and separate on plate from mixed culture, then check purity by gram stain

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11
Q

Why is isolation important in the study of bacteria?

A

we are able to identify and study the specific characteristics of the organisms, which could lead to cures for diseases

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12
Q

How is pasteurization of soil considered an enrichment culture?

A

Heat kills vegetative cells and leaves spores

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13
Q

What is the purpose of pour plate technique?

A

create lawns of bacteria, can be used to enumerate bacteria from the sample. Also can be used to check effectiveness of particular agent with disc diffusion method

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14
Q

Would the colonies produced by a pure culture appear different on the surface versus the ones growing in the agar?

A

Possibly, growth is restricted in agar matrix, possibly environmentally low O2 in agar

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15
Q

Can you deduce that diff bac grow on the surface of agar vs inside agar?

A

Could be the same (ex. facultative anaerobe) would survive on and in agar surface but would look different cause a colony will grow bigger on sfc and not trapped by agar matrix

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16
Q

What is a potential use for creating lawns of bacteria?

A

Creating lawns of bacteria helps to assay a variety of characteristics. For example, create a lawn of bac and test it for the sensitivity to an antibiotic

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17
Q

How do you isolate Bacillus subtilis from a soil sample?

A

need to pasteurize to kill non-spore formers

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18
Q

Why is it important to incubate the plates upside down?

A

It prevents the condensation from dripping down onto the plated bacteria which could cause the colonies to run together or contaminate the plate

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19
Q

What us the purpose of streak plate technique?

Why is it important in the study of bacteria?

A

It’s an isolation technique to separate bacterial colonies far enough apart that they grow in isolated colonies in an effort to get a pure culture. This is important to study individual bacteria and its morphology. It can also be helpful in clinical settings to diagnose and determine antibiotic needed to treat infection.

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20
Q

What are the pros and cons of serial dilution

A

Pros–> useful to isolate microorganisms that is predominant in a mixed culture
Cons–> it doesn’t separate bacteria, overnight incubation is required

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21
Q

Pros and cons of streak plate method

A

Pros–> good for isolation of cultures into individual and separate colonies
Cons–> Can be used for quality but not quantity studies, amount of inoculum not measured, higher probability of contamination before isolation

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22
Q

Pros and cons of spread/pour plate

A

Pros–>good for quantitative purposes

Cons–>it allows the growth of many microbes and units, contamination can occur, microbes sensitive to heat will die

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23
Q

Pros and cons of enrichment culture

A

Pros–>useful for isolation of specific microorganism, especially one that is present in much smaller numbers
Cons–>it takes a series of transfers, time consuming, easily contaminated, not representative of numbers normally in environment

24
Q

Pros and cons of selective media

A

Pros–> contains agents that inhibit growth of unwanted organisms
Cons–>can’t get clear view of bacteria in a sample

25
Pros and cons of differential media
Pros-->contains agents that help differentiate between organisms Cons-->growth is slow and easily contaminated
26
How is pasteurized soil sample considered an enriched culture
It is considered an enriched culture because we are getting rid of some microorganisms that aren't capable of surviving the increased temperature, and allowing those that can survive to grow. These microorganisms are usually found in small numbers in the soil
27
List two reasons the pour plate technique is used
1. To determine the amount of microorganisms in a specimen | 2. To identify the microorganism for diagnostic purposes
28
Describe streak plate technique
1. Aseptically, pick up a loopful of inoculum from broth culture and streak a vertical line up and down on 1st section. 2. Then move the loop in a zig zag pattern on 1/3 of the plate. 3. Flame loop and cool. Then rotate plate 90°. Spread bacteria from1st area into 2nd area and spread in a zig zag pattern 4. Flame loop and cool. Rotate 90°, spread bacteria from 2nd area to 3rd area in same pattern. 5. Flame loop. Replace lid. Invert plate and incubate plate at designated temperature.
29
At what temperature do you pasteurize soil and for how long?
In a water bath of 80° C for 15 minutes
30
At what temperature should molten TSA deeps be when poured onto plate
60°C or baby bottle warm
31
What is the appropriate colony count range for plate counting
20 to 200 or 30 to 300
32
Standard formula to solve for #bacteria per ml or g
CFU÷ (total dilution used × amt plated)
33
If duplicate plates with same amount plates were done, which one do you use for plate counting
Average the counts together
34
How do you determine total dilution for dilution tubes
Multiply the individual dilution of the tube with the previous total dilution= total dilution of new tube
35
What is the dilution for an individual tube
Amount of specimen transferred ÷ (the amount of specimen transferred + amount already on tube)
36
How do you determine the amount plated
The amount of dilution used to make the particular pour plate or spread plate
37
How do make a lawn plate
Use empty plate to pour 1ml of sample and pour melted agar deep to create lawn of bacteria
38
How do the colonies growing on the surface of agar vs those growing inside the agar differ? Are we able to to determine if they are different bacteria?
The colonies growing inside are smaller than those on the surface. If they're on the surface they could be obligate aerobes, aerotolerant, facultative anaerobes, If they're in the liquid they are - obligate anaerobes, aerotolerant, or facultative anerobes We could only speculate what type of bacteria they are.
39
What is a pure culture?
A culture in which all organisms are descendants of the same organism
40
Why do we study pure cultures?
To identify microorganisms and study microbial concepts such as growth characteristics, pathogenicity, metabolism, and antibiotic suspectibility (diagnostic reasons and research)
41
What is contamination?
Presence of unwanted microorganisms
42
What are the three dilution methods commonly used for the isolation of bacteria?
Serial dilution, pour plate/spread plate, streak plate
43
What is done in the spread plate technique?
A small amount of the previous diluted specimen is spread over the surface of a solid medium using a spreading rod
44
Would colonies produced in a pure culture appear different on the surface vs the ones growing in the agar
The colonies inside the agar would look different. This is dependent on available oxygen and space. Also on the surface one is able to evaluate the characteristics of the colonies.
45
Will the isolated colonies always be in the last section on the streak plate?
No, sometimes they grow in the section before, but usually they are too clumped together there to be isolated
46
What is a disadvantage of the pour plate technique?
Sometimes bacteria will clump together and look like a single colony when there are actually multiple or they might not grow adequately due to lack of space and oxygen
47
What is a potential use for creating lawns of bacteria
They are useful because of their heavy growth. They can be used to test sensitivity to different substances such as disinfectants and/or antibiotics
48
Why was the loop flamed in between streaks during the streak plate procedure
This was done in effort to isolate the colonies a little more each time. If the loop was not flamed each time then proper isolation of colonies would not occur and there would be to many colonies, too close together, or on top of one another.
49
How would you go about isolating a microorganism that is predominant in a mixed culture
I would use the serial dilution method to dilute the culture to the point that the predominant microorganism would be the only one to survive while other microorganism became extinct. I would plate 0.1 ml and 1ml of the final dilution.
50
Why is it important to incubate the plates upside down
So you can prevent the condensation from dripping onto the surface of the agar. This will prevent contamination and also prevent the colonies from growing too close or on top of one another
51
Serial dilution
Mixed culture could be serially diluted to the point of extinction to obtain a single microorganism. Useful to isolate a predominant microorganism in a mixed culture
52
Streak plate method
A small amount of mixed culture could be streaked to obtain separation on a solid medium.
53
Can streak plate be used for counting
No
54
Before a mixed culture is spread or poured, what must he done 1st
Serial dilution
55
Does streak plate allow counting of bacteria to represent what's in the environment
No, because it kills bacteria when taking a loopful
56
What is the goal of isolation
To grow sample where colonies are far enough apart to be able to count
57
Advantage pour plate | Disadvantage
Advantage-->Can grow more colonies, count them, good for growing facultative bacteria Disadvantage-->can kill those not tolerable to heat, obligate aerobes might not do well inside agar

Microbiology LAB (10 decks)

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