CSIM 1.37 Laboratory Investigation and Identification of Bacteria Flashcards
Which conditions effect bacteria survival and vary between species?
- Temperature
- pH
- Oxygen availability
- Salinity
What is a halophile?
A bacterium which lives in and prefers salinity
What does halotolerant mean?
Prefers no salinity but can survive in it
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
1) Obligate aerobe - Neisseria
2) Facultative anaerobe - E. coli
3) Aerotolerant anaerobe - Streptococcus
4) Strict anaerobe - Clostridium
5) Microaerophile - Many bacteria
(IMG 99)
What is a culture medium? What are the common components of this?
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)
What are the types of culture medium?
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
What are the macronutrients needed by bacteria?
- Carbon
- Oxygen
- Nitrogen
- Phosphorous
What is chocolate agar?
Blood agar (type of enriched media) whereby the blood has been lysed by heat
Give an example of a selective, differential media
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)
What is a broth?
A liquid culture medium
Give an example of a differential enriched media
Blood agar (intact RBCs) • Differentiates between haemolytic and non-haemolytic bacteria
What are the types of haemolysis seen on blood agar plates?
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
What are the arrangements of bacteria in test tubes when grown with broth cultures?
- Pellicle (near top)
- Turbidity (all throughout)
- Sediment (near bottom)
IMG 100
What biochemical tests can be used to help identify bacteria?
- Catalase test
- Indole test
- Carbohydrate utilisation test
- Analytical profile index (API) test
- Antibiotic susceptibility testing
Describe the catalase test
• 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
How can you differentiate between Staphylococcus and Streptococcus?
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)
What is the indole test
• 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)
Describe the carbohydrate utilisation test
- 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
Describe an API test
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
Describe an antibiotic susceptibility test
- 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
What does a ‘blurry’ line mean when around a zone of inhibition?
It means that at that particular concentration the antibiotic is bacteriostatic rather than bacteriocidal
What does dots within an inhibition mean?
Resistant mutants (IMG 104)
What is an antibiotic strip?
A strip with a range of concentrations for a certain antibiotic to see at which concentration of antibiotic sensitivity begins (IMG 105)