CSIM 1.37 Laboratory Investigation and Identification of Bacteria

Question 1

Which conditions effect bacteria survival and vary between species?

Answer 1

• Temperature
  
  • pH
  • Oxygen availability
  • Salinity

Question 2

What is a halophile?

Answer 2

A bacterium which lives in and prefers salinity

Question 3

What does halotolerant mean?

Answer 3

Prefers no salinity but can survive in it

Question 4

What are bacteria referred to as if:

1) They require oxygen
2) They prefer oxygen
3) They ignore oxygen
4) Oxygen is toxic
5) Require oxygen, but are poisoned by high concentrations of oxygen (2-10% needed)

Give an example of each

Answer 4

1) Obligate aerobe - Neisseria
2) Facultative anaerobe - E. coli
3) Aerotolerant anaerobe - Streptococcus
4) Strict anaerobe - Clostridium
5) Microaerophile - Many bacteria

(IMG 99)

Question 5

What is a culture medium? What are the common components of this?

Answer 5

A mixture of nutrients devised to support the reproduction of microorganisms
• Peptones (protein hydrolysates prepared by partial digestion)
• Extracts (beef or yeast)
• Agar (polysaccharide used to solidify liquid media)

Question 6

What are the types of culture medium?

Answer 6

General purpose media
• Support the growth of many microorganisms

Enriched media
• General purpose media supplemented by blood or other special nutrients

Solid selective media
• Favours the growth of some organisms and inhibits the growth of others

Solid differential media
• Distinguish between different groups of microorganisms based on biological characteristics

Question 7

What are the macronutrients needed by bacteria?

Answer 7

• Carbon
  
  • Oxygen
  • Nitrogen
  • Phosphorous

Question 8

What is chocolate agar?

Answer 8

Blood agar (type of enriched media) whereby the blood has been lysed by heat

Question 9

Give an example of a selective, differential media

Answer 9

MacConkey agar which contains bile salts
• Selective for gram-negative enteric bacteria (e.g. E. coli)
• Differential because lactose fermenters will turn the dye red, whereas lactose nonfermenters will not (IMG 98)

Question 10

What is a broth?

Answer 10

A liquid culture medium

Question 11

Give an example of a differential enriched media

Answer 11

Blood agar (intact RBCs)
  •  Differentiates between haemolytic and non-haemolytic bacteria

Question 12

What are the types of haemolysis seen on blood agar plates?

Answer 12

α-haemolysis
• Partly breaks down the RBCs and uses the iron, creating a green-ish discolouration from the red blood

β-haemolysis
• Produces TOTAL haemolysis which turns the red blood cells into completely clear agar

𝛾-haemolysis
• No haemolysis and no change in the blood agar

IMG 97

Question 13

What are the arrangements of bacteria in test tubes when grown with broth cultures?

Answer 13

• Pellicle (near top)
  
  • Turbidity (all throughout)
  • Sediment (near bottom)

IMG 100

Question 14

What biochemical tests can be used to help identify bacteria?

Answer 14

• Catalase test
  
  • Indole test
  • Carbohydrate utilisation test
  • Analytical profile index (API) test
  • Antibiotic susceptibility testing

Question 15

Describe the catalase test

Answer 15

• Catalase is an enzyme which carries out the stabilisation of superoxide molecules:

2H₂O₂ → 2H₂O + O₂

• Take a plate, add hydrogen peroxide, and see if effervescence occurs
• If it does, this suggests oxygen is being made and that catalase is present

Question 16

How can you differentiate between Staphylococcus and Streptococcus?

Answer 16

Staphalococcus and Streptococcus:
• Both Gram positive
• Both cocci
• Bothe grow in air

HOWEVER, Staphylococcus uses respiration and is catalase positive, whereas Streptococcus uses fermentation and is catalase negative

Therefore the catalase test can be used, positive = Staphylococcus (IMG 101)

Question 17

What is the indole test

Answer 17

• Tests for the enzyme trytophanase:

L-tryptophan + H2O -> indole + pyruvate + NH3

• Indole has a red colour if mixed with KOVAC’S REAGENT
• Kovac’s reagent does not turn red if indole from trytophanase activity is present (IMG 102)

Question 18

Describe the carbohydrate utilisation test

Answer 18

• A Durham tube is used (test tube upside-down in another test tube with broth and bacteria)
  
  • Gas produced by bacteria in the inverted test tube is collected
  • Results can be negative (no gas produced) doubtful (small amount of gas produced) or positive (condiferable amount of gas produced)
  • This co-incides with a decrease in pH

Question 19

Describe an API test

Answer 19

Bacteria are placed into numerous enclosed containers with the compositions needed for specific biochemical tests. The results for each are ran by a database to identify the bacteria species

Question 20

Describe an antibiotic susceptibility test

Answer 20

• A nutrient agar plate is inoculated with a culture
  
  • Small discs coated in different antibiotics are placed onto the surface of this agar plate
  • The organism will not grow around antibiotics it is sensitive to creating ‘zones of inhibition’

IMG 103

Question 21

What does a ‘blurry’ line mean when around a zone of inhibition?

Answer 21

It means that at that particular concentration the antibiotic is bacteriostatic rather than bacteriocidal

Question 22

What does dots within an inhibition mean?

Answer 22

Resistant mutants (IMG 104)

Question 23

What is an antibiotic strip?

Answer 23

A strip with a range of concentrations for a certain antibiotic to see at which concentration of antibiotic sensitivity begins (IMG 105)