Lab 2 Flashcards
1
Q
What are biochemical profiles (called Biotypes)?
A
- They are based on differences in biochemical reactions, morphology, and environmental tolerances (e.g., ability to grow at a given pH or temperature).
- Identification depends on these biotypes, and this is tested by inoculating different types of selective or differential media.
2
Q
What general observations of media should be made post-incubation? [3]
A
- Colour changes
- Contamination
- Colony or growth morphology (a.k.a. macroscopic morphology)
3
Q
Describe various contamination types post-incubation of media. [4]
A
- Airborne contamination: observe the plate for any colonies that are not on the streak lines.
- Non-aseptic techniques in pouring of the plate: look for colonies within the medium.
- Contamination of the loop: look for colonies on the streak lines at the beginning of the plate that appear different from the colonies on the rest of the plate.
- Surface contamination: look for a line of different colonies occurring where the contaminant has been picked up on the loop.
4
Q
Desribe how to differentiate between colony growth and non-colonial growth post-incubation.
A
- Colonies - separated cells on an agar plate results in isolated colonies following incubation. Seven characteristics are used to describe a colony on an agar plate.
- Growth (non-colonial) – a smear of growth can be described merely by using color and consistency.
5
Q
Describe how liquid growth post-incubation is described. [3]
A
- Negative → solution is transparent → no growth
- Weak positive → little growth, liquid is slightly turbid
- Strong positive → lots of growth, liquid is very turbid/cloudy
6
Q
Describe Luria Agar.
A
- Supports growth of a variety of organisms due to various nutritional components it provides.
7
Q
Describe MacConkey Agar.
A
- Used for isolation and detection of Gram-negative bacteria rods like Salmonella, Shigella, and Escherichia from stool, urine, water, or food samples.
- The crystal violet and bile salts selectively inhibit the growth of many Gram-positive organisms and promote the growth of the Gram-negative rods.
- Lactose is included in the medium.
- Allows for the differentiation between lactose positive and lactose negative organisms.
8
Q
How do Gram-negative rods that ferment lactose appear on MacConkey Agar?
A
- These microbes ferment lactose and produce acid, which causes the pH indicator (neutral red) to change.
- Red colonies result due to the neutral red dye, which is colorless above pH 6.8 and red below pH 6.8.
- As a result of the low pH, a zone of precipitated bile may surround the colonies.
9
Q
How does Enterobacter appear on MacConkey agar?
A
- This is a lactose positive microbe.
- This organism uses the lactose rapidly and colonies appear pink (vs. red) with light-pink to white centers.
- When lactose is completely utilized, the organism begins to metabolize other components in the media and change the pH indicator again.
10
Q
How do Salmonella and Shigella appear on MacConkey agar?
A
- These microbes are lactose negative.
- They do not change the pH indicator and will appear as amber, translucent colonies.
11
Q
Describe Eosin MethyleneBlue (EMB) agar.
A
- The two dyes in EMB or Eosin Y and methylene blue agar plates inhibit the growth of gram positive organisms and, along with the lactose in the medium, allow for differentiation of enteric bacteria based on the colony morphology.
- Typical fecal coliform (e.g., E. coli) colonies are small, dark red and nucleated with a green metallic sheen due to acidic pH. The green metallic sheen is indicative of vigorous lactose fermentation.
- Non-fecal coliforms (e.g., E. aerogenes) may have larger, pink, possibly mucoid, nucleated or unnucleated colonies lacking a metallic sheen due to smaller amounts of acid production due to slow fermentation of the sugars.
- Non-fermenters remain colorless - or the color of the medium.
12
Q
Describe Xylose-lysine-deoxycholate (XLD) agar.
A
- As implied by the name, this medium contains xylose, L-lysine, sodium deoxycholate, as well as ferric ammonium citrate.
- Sodium deoxycholate will inhibit Gram-positives.
- Acid or alkaline byproducts are detected by a pH indicator, Phenol Red, which is yellow at acidic pH and red/pink at alkaline pH.
- Fermentation of xylose is indicated by yellow colonies due to acidification of the medium.
- The L-lysine, an amino acid, is provided for decarboxylation reactions.
- Organisms that can decarboxylate lysine will release alkaline by-products and produce red colonies.
- Ferric ammonium citrate acts as an indicator of the presence of sulfide gas (H2S) because of sulfur reduction.
- A black precipitate will form on the growth due to the reaction of ferric ammonium citrate with H2S.
13
Q
Describe how bacteria is visualized.
A
Macroscopy: color; shape; border
Microscopy: gram; shape; arrangement; spores?
14
Q
Describe how to make a suspension.
A
- Disinfect working surface
- Turn burner on
- Sterilize loop by flaming
- Allow loop to cool
- Transfer bacteria
- Flame loop again
15
Q
When inoculating media, when do we use a loop versus a needle?
A
16
Q
Give 5 examples of biochemical tests.
A
- Hydrolysis
- Carbohydrate utilization
- Amino acid degradation
- Respiration test
- Selective and/or differential media
17
Q
Describe how hydrolysis can be used as a biochemical test.
A
- Test the ability of some bacteria to hydrolyze a particular compound
- E.g., casein hydrolysis
18
Q
Describe how carbohydrate utilization is used as a biochemical test.
A
- Test the ability of some bacteria to ferment a particular CHO
- Make a broth with the CHO that we want to test and add a pH indicator and a Durham tube (a mini test tube that is put in upside down)
19
Q
Describe how amino acid degradation can be used as a biochemical test.
A
- Test the ability of some bacteria to degrade a particular amino acid
20
Q
Describe how respiration tests can be used as a biochemical test.
A
- Catalase test: detect the presence of catalase, an enzyme that degrades hydrogen peroxide.
23
Q
Describe Eosin Methylene blue (EMB) agar.
A
- Eosin Y and methylene blue inhibit the growth of Gram-positive organisms and are also pH indicators.
- Green and black colonies are lactose fermenters
- Colourless or pink colonies are not lactose fermenters
- Selective and differential media
27
Q
Describe the Enterobacteriaceae family. [4]
A
- Usually comprised of bacteria that inhabit the human intestinal tract.
- 44 Genera - Escherichia, Salmonella etc.
- 170+ species – E. coli, S. typhi (in latest edition of Bergey’s) – with more to be added!
- Chemoorganotrophs – use organic sources for energy, electrons and carbon e.g., sugars such as glucose. Can perform aerobic and anaerobic cellular respiration AND fermentation. Species differentiation based on biochemical tests can be done using Table 3 in Bergey’s manual.
29
Q
What are the eight characteristics used to describe bacterial colonies on agar plates?
A
- Size (diameter in mm)
- Shape (whole colony; round; irregular; filamentous; spindle)
- Margin (outer edge of colony; smooth; wavy; filamentous)
- Pigmentation (colour)
- Texture (smooth; wrinkled; rough; concentric rings)
- Appearance (matte or shiny)
- Optical properties (translucent or opaque)
- Elevation (flat; raised; convex; umbonate; craterform)
30
Q
Describe MacConkey agar.
A
- Key ingredients are crystal violet/bile salts and lactose/neutral red.
- Crystal violet/bile salts inhibit growth of gram-positive bacteria
- Lactose/neutral red - bacteria that can ferment lactose produce acid and change the pH.
32
Q
Describe EMB agar.
A
- Key ingredients are eosin Y/methylene blue and lactose
- Eosin Y/methylene blue – inhibits growth of Gram-positive bacteria
- Eosin Y/methylene blue/lactose - bacteria that can ferment lactose produce acid which change the pH resulting in the green metallic sheen
33
Q
Describe XLD agar.
Key ingredients and what they do
A
- Key ingredients are (1) xylose, (2) L-lysine, and (3) sodium deoxycholate
- Sodium deoxycholate – inhibits growth of Gram-positive bacteria
- Xylose – a sugar that can be fermented, resulting in pH change in media (yellow colonies) (right)
- L-lysine – an amino acid that can be decarboxylated (a carboxyl group is removed and released as CO2), resulting in pH change in media (red colonies) (left)
34
Q
What does an indole motility tube test for? [3]
A
- The ability of an organism to:
- Swim through the agar (growth will radiate from the stab line and the tube becomes turbid) (2nd from left).
- Reduce sulfur to H2S, resulting in a black colour forming (right).
- Produce indole from the amino acid tryptophane (detect by adding Kovac’s reagent - (2nd from left).
35
Q
What does inoculation of Durham tubes with glucose/lactose/sucrose broths test for?
A
- An organism’s ability to ferment the sugar and produce gas
- This is a differential test
36
Q
What does inoculation of MRVP broths test after growth? [2]
A
- Methyl red test - to detect the ability of an organism to produce stable acids end products (mixed-acid fermentation) (top).
- Voges Proskauer – to detect the ability to produce acetylmethyl carbinol (bottom).
37
Q
What does innoculation of nitrate broths with Durham tubes test for?
A
- It tests the ability of an organism to reduce nitrate (NO3–) to nitrite (NO2–) or N2 (nitrogen gas).
- Nitrite is detected by adding zinc dust after growth.
- Nitrogen gas is detected in the Durham tube.
38
Q
What does the urea slant test for?
A
Ability to split urea to ammonium and CO2.
39
Q
What does the citrate slant test for?
A
Ability to use citrate as an energy source.
40
Q
What does the bile esculin slant test for?
A
Ability to use esculin as an energy source in the presence of bile
