LAB 6

Question 1

What is the incidence of salmonellosis gastroenteritis

Answer 1

An estimated 1.35 million cases occur annually
- 26,500 result in hospitalization
- 420 result in death

Question 2

symptoms of salmonellosis gastro

Answer 2

• Abdominal pain
• Cramps
• Diarrhea
• Nausea
• Vomiting
• Fever
• Dehydration

Question 3

metagenomics def

Answer 3

The study of microbial genomes based on DNA extracted directly from the environment, it samples the entire pool of nucleic acids found in any given ecosystem and most frequently used to determine the members of the microbial community living there

Question 4

metatranscriptomics def

Answer 4

mRNA extracted from the natural environment is reverse transcribed to cDNA and directly analyzed by next generation sequencing

Question 5

what is the incidence of nasal carriage of s.aureus in the MICR221 class

Answer 5

31%

Question 6

results of TSA

Answer 6

Enteroccus faecalis
escherichia coli
salmonella typhimurium
shigella sonnei
All had:
- 0.5 mm
- raised
- circular
- entire
- cream
- smooth

Question 7

hektoen agar

Answer 7

• an example of selective and differential medium used for the isolation and cultivation of gram-negative enteric microorganisms

Question 8

Enterococcus faecalis hektoen agar results

Answer 8

growth = complete inhibition

Question 9

Escherichia coli hektoen agar results

Answer 9

growth = partially inhibited
colony morphology = orange-red, smooth, raised

agar colour = orange

Question 10

salmonella typhimurium hektoen agar results

Answer 10

growth = good
colony morphology = 2-4mm, clear, green/blue, moist, raised, black centres
agar colour = blue/green

Question 11

shigella sonnei

Answer 11

growth = good
colony morphology = 2-3mm, clear, green/blue, moist, flat, transparent
agar colour = blue/green

Question 12

selective components in hektoen agar

Answer 12

bile salts

Question 13

which group of bacteria is hektoen agar selective for

Answer 13

enteric bacteria

Question 14

differential components in hektoen agar

Answer 14

Lactose
Sucrose
Salicin
Acid fuchsin
Bromothyl blue
Ferric ammonium citrate
Sodium thiosulfate

Question 15

how does hektoen agar differentiate between the groups of bacteria

Answer 15

Salmonella produce hydrogen sulfide producing black colonies, other enteric bacteria (including Shigella) do not

Salmonella and Shigella do not ferment any of the three carbohydrates (lactose, sucrose or salicin) so the agar colour stays the same (blue/green), unlike other enteric bacteria (salmon orange-red)

Question 16

which enzyme must bacteria have in order to grow in tetrathionate broth

Answer 16

tetrathionate reductase

Question 17

why does salmonella need to be incubated on an enrichment broth

Answer 17

• because it is present in such small numbers it may be impossible to recover
• the tetrathionate broth allows the salmonella to multiply and also suppresses growth of the normal faecal microflora

Question 18

results of enriching salmonella

Answer 18

Primary culture (inoculated with a faecal sample):
colony morphology:
- orange colour
- smooth
- raised
agar colour:
- orange + may be a precipitate

Enrichment subculture:
colony morphology:
- 2-4mm
- clear
- green
- moist
- raised
- black centres
agar colour:
- mostly green/yellow

Question 19

estimate for what the bacteria growing on the primary culture plate in the enrichment of salmonella is

Answer 19

E.coli

Question 20

estimate for what the bacteria growing on the enrichment culture plate in the enrichment of salmonella is

Answer 20

Salmonella typhimurium

Question 21

triple sugar iron agar

Answer 21

• differential but not selective
• useful in screening isolates of enteric bacteria
• contains three carbs:
  -> glucose
  -> lactose
  -> sucrose
• also contains
• ferrous ions
• pH indicator phenol red

Question 22

what are the four reactions that can be recorded from each tube in the triple sugar ion agar

Answer 22

• colour of the butt
• colour of the slant
• production of gas
• production of hydrogen sulfide

Question 23

what are the possible outcomes of the triple sugar iron agar

Answer 23

1. Acid reaction (yellow colour) = indicates the ability of the bacteria to ferment one or more of the carb components of the medium
2. Alkaline reaction (deep red colour) = indicates the aerobic utilisation of organic acids by enteric bacteria when the fermentable substrates have been utilised
3. sulfide produced by the bacteria reacts with ferrous ions in the medium producing insoluble ferrous sulfide (black)
4. gas production can be recorded as positive if gas bubbles in the agar or at the base of the tube are observed

Question 24

triple sugar iron agar test results

Answer 24

Escherichia coli:
slant = yellow
butt = yellow
gas = yes
H2S = no
Proteus mirabilis:
slant = red
butt = yellow
gas = yes
H2S =yes
Salmonella:
slant = red
butt = yellow
gas = yes
H2S =either
shigella sonnei:
slant = red
butt = yellow
gas = no
H2S =no

Question 25

what organisms could be confused using TSI slants

Answer 25

Proteus mirabilis and Salmonella

Question 26

urease test

Answer 26

- used when organisms cannot be differentiated by TSI slants - differential - used to test the ability of an organism to produce the exo-enzyme, urease - urease breaks the carbon-nitrogen bond of amides to form CO2, ammonia and water Contains two buffers, urea, a very small amount of nutrients for the bacteria, agar, and the pH indicator phenol red --> if the urea in the slant is degraded and ammonia is produced, an alkaline environment is created and the media turns pink

Question 27

results of the urease test

Answer 27

proteus mirabilis = pink = alkaline (contains urease) salmonella = yellow = acidic (no urease)

Question 28

carcinogenic

Answer 28

having the potential to cause cancer

Question 29

colony morphology of the enteric bacteria

Answer 29

E.coli & S.typhimurium - gram negative - regular staining - rods - singles - 2um - regular form - straight rod axis --> therefore they can't be differentiated from the gram stain alone

Question 30

serotype

Answer 30

a way of grouping cells based on the antigens or other molecules found on their surface

Question 31

identification of Salmonella species

Answer 31

- a serotype is determined by the somatic (O) and flagella (H) antigens in the cell wall of Salmonella

Question 32

nomenclature for identification of salmonella species

Answer 32

- in antigenic formulae the O is listed first then the H - major antigens are separated by colons and the components of an antigen (serofactors) are separated by commas eg: 4,5,12: j: 1, 2

Question 33

the slide agglutination test

Answer 33

- a rapid diagnostic test for typhoid fever - rapid results - used to test unknown organisms against known antisera positive result = clumping

Question 34

antisera

Answer 34

a blood serum containing antibodies

Question 35

the slide agglutination test procedure

Answer 35

1. Place one 10 µL drop of the killed Salmonella isolate culture at each end of a microscope slide. 2. Add 10 µL of O antiserum 4 to one drop. 3. Add 10 µL of O antiserum 9 to the other. 4. Mix the drops by gently 'rocking' the slide. The drops need to stay separate so do not be too vigorous. Repeat steps 1 - 4 using a fresh slide with H antigen 1 and H antigen 5.

Question 36

what was the outcome of the slide agglutination test

Answer 36

typhimurium

Question 37

what is the gram reaction of C.difficile

Answer 37

gram positive

Question 38

what causes the symptoms of pseudomembranous colitis

Answer 38

--> Toxin A and Toxin B - the disease is not an infection as such, but rather an overgrowth of Cl. difficile in the bowel - this is due to the prior antibiotic therapy removing elements of the normal microflora, which usually keep this organism under control

Question 39

what types of antibiotics are most often associated with this condition?

Answer 39

- ampicillin - cephalosporins - clindamycin - amoxicillin - fluoroquinolone

Question 40

where is the largest bacterial community inhaibting the human body found

Answer 40

in the large intestine

Question 41

polymerase chain reaction (PCR)

Answer 41

- DNA or RNA is extracted directly from intestinal or faecal samples - then the hypervariable 16S rRNA sequences are amplified using PCR primers that anneal with the conserved sequences that span selected V regions - to seperate the components 16S fragments in the PCR product, a polyacrylamide gel is used - The double-stranded 16S fragments migrate through a polyacrylamide gel containing a gradient of urea and formamide until they are partially denatured by the chemical conditions - Due to the variation in the 16S sequences of different bacterial species, different 16S 'species' can be differentiated by this electrophoretic method.

Question 42

what prevents denaturation of the DNA gramgents during gel electrophoresis

Answer 42

a GC-right 5' end

Question 43

which group of bacteria predominate in babies and why is this so?

Answer 43

Bifidobacteria - the carbohydrate in breast milk (Bifidus factor) favours their growth

Question 44

which group of bacteria predominate in adults and why is this so?

Answer 44

Clostridium - due to diet, bifidobacteria are in the minority in the adult gut

Question 45

aim of the human microbiome project

Answer 45

has the goal of identifying and characterizing the microorganisms which are found in association with both healthy and diseased humans

Question 46

what were the two sites that the researchers noted as being "especially diverse in terms of community membership"

Answer 46

- oral and stool sites

Question 47

What is the site that the researchers noted as “harbouring particularly simple communities”?

Answer 47

vaginal sites

Question 48

name the dominant taxa in the oral cavity

Answer 48

streptococcus

Question 49

what were the percentage of S. aureus and S. epidermis found on participants skin and nasal samples

Answer 49

Skin Nasal S. aureus 4% 29% S. epidermidis 100% 93%

Question 50

thermal death point (TDP)

Answer 50

the lowest temp required to kill all bacteria in a sample within a period of 10mins

Question 51

thermal death time

Answer 51

the length of time required to kill all bacteria in a sample at a given temp

Question 52

decimal reduction time

Answer 52

the time taken to obtain a 10-fold reduction in the number of bacteria

Question 53

bacillus subtilis thermal death point

Answer 53

100 degrees

Question 54

escherichia coli thermal death point

Answer 54

70 degrees

Question 55

why does B. subtilis have a higher thermal death point

Answer 55

B. subtilis is more heat resistant as it forms endospores