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Lab 11 Flashcards

Bacterial conjugation

1
Q

Bacterial asexual reproduction

A

Bacterial reproduction by splitting of cells through binary fission. This transfer of genes from the parent cell to the daughter cell (one generation to the next) is a vertical transfer. They are basically identical copies or clones of the parent cell.

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2
Q

Does binary fission allow for genetic diversity?

A

No, the daughter cells are identical copies of the parent cell. This would only happen if a random mutation occurred, which occasionally happens

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3
Q

What are other ways the transfer of genetic information occurs in bacteria ?
Does this increase genetic diversity?

A

Genetic information can be transferred laterally. The transfer occurs from a donor cell to a recipient cell. This can occur by transformation, transduction, or conjugation.
This can increase genetic diversity.

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4
Q

What information can be transferred to recipient cells after lateral transfer has occurred

A

Transfer of genetic information may confer the recipient bacteria what traits they did not possess before the transfer occured. This includes virulence factors, resistance factors, metabolic traits. These can increase their chances of survival in a dynamic environment.

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5
Q

Transformation

A

A bacterium takes up a piece of (naked) DNA floating in its environment that was shed by other bacteria.

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6
Q

What resistance factors can a bacteria gain from transformation

A

Resistance to heavy metals or antibiotics

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7
Q

How can additional metabolic traits help bacteria

A

It can allow them to break down substances they were not able to before or inactive certain drugs or toxins

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8
Q

What did lab 11 show

A

2 different strains of E.coli were used, a donor F+ with a fertility plasmid with Ampicillin antibiotic resistance gene on it and a recipient or F- that lacked the fertility plasmid but had a gene for resistance to Streptomycin antibiotic on its chromosomal DNA. It was evident that the transfer of genetic information occured because growth occurred on an agar plate containing both Streptomycin and Ampicillin. The F+ bacteria transferred its Ampicillin resistance to the F- bacteria. The F+ bacteria died due to its lack of resistance to Streptomycin. The F- bacteria survived and created the lawn because it is now resistant to Ampicillin and Streptomycin. Recombination occurred and F- bacteria is now F+ with a fertility plasmid.

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9
Q

Plasmid

A

An extrachromosomal circular piece of double stranded DNA that has it’s own replication site. Genotypes can make phenotypes.

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10
Q

What does a F factor code carry ? How are they identified?

A

Codes for a pilus found on the surface of many bacteria. F+ signifies fertility factor. F+ strains are identified as possessing the fertility plasmid. F- strains lack it.

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11
Q

How does bacterial conjugation occur

A
  1. Cell to cell contact must occur:
    Gram- bacteria–> from donor to recipient via conjugation pilus
    Gram+–> less common,direct contact, cell to cell with sticky surface molecules/proteins, no pilus
  2. Conjugating cells are of opposite mating type
  3. Plasmid is replicated during transfer of a single stranded copy of the plasmid DNA to the recipient, complementary strand is replicated.
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12
Q

Pilus

A

A hair like appendage found on the surface of bacteria. It can act as a bridge between 2 strains of bacteria for transfer of genetic material.

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13
Q

When a F factor is transferred from a donor F+ to a recipient F-, the F- cell is converted to

A

F+ cell

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14
Q

Genetic recombination

A

The exchange of genes between 2 DNA molecules to form new combinations of genes in a chromosome.

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15
Q

Recombinant

A

A recipient cell that has incorporated donor DNA into it’s own DNA

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16
Q

When F factor becomes integrated into the chromosome of an F+ cell, it makes the cell

A

A high frequency of recombination cell (Hfr). Recombination between F factor and chromosome occur at a specific site.

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17
Q

When an Hfr donor passes a portion of its chromosome to an F- recipient

A

A recombinant F- cell results. The chromosome usually breaks before it is completely transferred and only a portion of the F factor from the Hfr cell combines with the recipient DNA. The rest is degraded. The F- acquires new versions of chromosomal genes but not a complete F factor and therefore maintains its F-.

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18
Q

R factor

A

Resistance factor located on plasmids

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19
Q

F’

A

F prime. When theF plasmid inaccurately excised from the chromosome after formation of an Hfr, it can take a portion of the bacterial chromosome with it. This then becomes part of thethe plasmid itself. This form of theF plasmidis called anF’.
(F+–>Hfr–>F’)

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20
Q

F+

A

Bacterial cell with a plasmid with a fertility factor

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21
Q

F-

A

Bacterial cell with no plasmid or fertility factor

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22
Q

F’

A

A bacterial cell with part of its chromosome DNA integrated into its plasmid

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23
Q

Hfr

A

Bacterial cell with its plasmid is now part of its chromosomal DNA

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24
Q

F’ + F(-)–>

A

F’

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25
Why dont we test for pathogenic bacteria in water
It's time consuming, a slow process, requires a lot of work, expensive, low concentrations are hard to detect, they include a diverse population of microorganisms, don't survive long enough be tested
26
What are good indicators of fecal contamination
Escherichia coli and Entercoccus faecalis
27
Why are Escherichia coli and Entercoccus faecalis good indicators of fecal pollution
They are always found in intestines, they're not normally in soil and water, easy to identify, survive longer than enteric pathogens
28
Do Escherichia coli and Entercoccus faecalis stay in the water a long time
No, they are non spore forming bacteria, so their survival in water is not extensive
29
What are the differences between Escherichia coli and Entercoccus faecalis ? Alike?
1. Escherichia coli--> gram negative,rod shaped cells, coliform 2. Entercoccus faecalis --> gram+, cocci cells, entercoccus 3. Alike in that non spore forming, found in GI tract
30
Coliform
An aerobe or facultative anaerobe that ferments lactose to produce C02 gas, gram negative, non spore forming, rod shaped cells, nonpathogenic
31
What bacteria are Coliforms
E. coli and Enterobacter aerogenes
32
Unacceptable level of e.coli in marine water
> 126 e.coli per 100mL
33
What is the Compact Dry EC method
It is a ready to use chromogenic medium used for performing E.coli and coliform counts that contain dehydrated culture media and cold water-soluble gelling agent in a non woven cloth matrix
34
How does Compact Dry EC method work
The medium is instantly hydrated when inoculated with a sample and capillary action difuses the sample evenly over the matrix to form a gel within seconds. The compact dry method contains 2 chlorogenic substrates, Magenta-Gal and X-Gluc that yield a colored reaction and allow differentiation of E.coli from other members of the coliform group.
35
How does E.coli produce a different result from other coliforms in the Compact Dry method
E.coli produces the enzyme B-glucuronidase, it reacts with X-gluc to form blue colonies
36
What do the different colors on the Compact Dry method mean
Pink-purple colonies--> other coliforms Blue colonies-->E.coli Colorless or yellow-->Pseudomonas spp
37
How much water was inoculated into Dry Compact method
1mL
38
What is involved in the Most Probable Method
3 different tests: 1. Presumptive Test 2. Confirmed Test 3. Completed Test
39
Presumptive test
A series of 9 to 12 tubes of lactose broth are inoculated with measured amounts of water; 10mL, 1.0mL, 0.1mL. and incubate at 35°C for 24 hours. If the water contains any lactose fermenting bacteria it will produce gas (10% or more). It is presumed that coliforms are present. The test is also used to determine the MPN.
40
How do you determine the MPN and what does it mean
You use the series of tubes that were inoculated, the first series of 3, second series of 3, and 3 series of 3. How many of the first series showed gas, how many in the 2nd and 3rd. Those numbers are compared to the MPN table. The number on the chart represents the amount of organisms/ coliforms per 100mL of the sample
41
Is MPN an accurate representation
It is an approximation and has a 95% probability of being within a specific range. It is a statistical probability figure.
42
Confirmed test
Plates of Levine EMB agar are inoculated from positive, gas producing tubes to see if the organisms that are producing gas are gram negative.
43
What is EMB
Eosin, methylene blue (6:1), and lactose sugar agar. It is a selective and differential medium. Methylene blue and eosin to a lesser extent inhibits the growth of gram positive bacteria. Eosin changes color to a dark purple when the medium becomes acidic indicating lactose fermentation. This allows colonies of coliform to be distinguished from non coliforms since coliforms are lactose fermenters.
44
Why dont we just assume unsafe water due to coliforms present in the water sample since the presumptive test showed gas indicating lactose fermentation
Gas formation could be due to non coliform bacteria such as Clostridium perfringes that are gram positive
45
What is the purpose of EMB and Mac in the confirmed test
To confirm the presence of gram negative, lactose fermenting bacteria
46
How can you tell if E.coli is growing on EMB
They produce small dark colonies usually with a distinct metallic green sheen
47
What can grow on EMB plate that is not an indicator of fecal pollution? Why is it not a good fecal indicator?What does it look like?
1.Enterobacter aerogenes 2. It is also found in soil and grains 3. Large colonies with dark centers and lighter rims ( fermenters)
48
Mac
MacConkey agar plate. It is selective and differential. It has crystal violet and bile salts that inhibit gram positive bacteria and allows the growth of gram negative bacteria. Enteric bacteria ferment lactose and is indicated with the carbohydrate lactose and neutral red pH indicator. It turns red when pH drops below 6.8 *fermentation)and colorless above this.
49
What must we be sure to use in the confirmed portion of the MPN method
Presumptive tubes that were positive for gas production are streaked on EMB and Mac plates and incubated at 37°C for 24 hours
50
Completed test
This test is to determine if the isolate from the agar plates truly matches the definition of a coliform. The media used is a TSA slant and lactose broth with an inverted Durham tube incubated at 35°C. If gas is produced and a slide from the agar slant reveals gram negative, non spore forming rod cells, then we can be certain they are coliforms.
51
After the completion of the 3 tests in the MPN method, can we establish that the water is safe or unsafe
No, we have established that there were coliforms present, but we don't know if they are E.coli or E.aerogenes (which can be found in soil). Therefore we have to perform IMViC tests to differentiate between the 2 species.
52
Eneterobacteriaceae
Gram negative, non endospore forming rods, oxidase negative, glucose fermenters and nitrate reducers, can be coliforms (nonpathogenic) or noncoliforms (pathogenic)
53
Noncoliforms
Lactose non fermenters, pathogenic
54
IMViC
Biochemical test involving Indole, Methyl red, Vosges-proskauer, Citrate
55
Pros of MPN
1. Uniform recovery of a microbial population; taking a large sample size and converting to 100mL 2. Realistic representation of count 3. Growth of bacteria unculturable on agar
56
Cons of MPN
1. Labor and materials intensive 2. It takes 3 days to complete 3. Requires more time for identification 4. MPN estimates often have a lower order of precision
57
Pros of Compact Dry Method
1. Easy to use 2. No preparation required 3. Highly portable and can be stored up to 18 months 4. Relatively inexpensive 5. Convenient 6. Very specific and selective
58
Cons of Compact Dry Method
1. Maybe considered too expensive if a lot of plates are required to determine a more accurate number 2. Can be difficult to count colonies with high coliform or E.coli counts 3. Cannot distinguish 0157:H7 E.coli strain because doesn't produce B-glucuronidase
59
Magenta-Gal
Has lactose and glucose, targets lactose fermenters. It is cleaved by B-galactosidase to produce purple or pink-->reddish
60
X-gluc
Is cleaved by B-glucuronidase to produce blue, exclusive to E.coli

Microbiology LAB (10 decks)

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