Microbio Pracs Flashcards
AA. LEARNING OUTCOMES
- Understand the basis on which bacteria are differentiated & classified.
- Be familiar with the application of one method, that of Cowan & Steel, for the identification of medically important bacteria.
- Understand the basis of some biochemical, rapid & molecular methods currently available for lab identification of medically implicated microbes.
- Be able to apply these methods to characterise & identify micro-organisms.
- Have a significant understanding of the importance to human health & well-being of the relationship bw microbes & human host defences.
AB. Morphological characterisation of bacteria involves assessment of:
A. results from cultural tests
B. biochemical tests for metabolic pathways
C. serological tests for presence of bacterial antigens
D. PCR probing and amplification for presence of specific sections of DNA
A. results from cultural tests
AC. Biochemical tests are used to determine:
A. production of immunoglobulins
B. substrate utilisation
C. serological characteristics
D. DNA base sequences
B. substrate utilisation
AC. Name 6 characteristics used to describe bacterial colony morphologies on solid media.
SIZE - measured as diameter in mm
SHAPE - e.g. circular, filamentous, irrregular, punctiform, spindle, rhizoid
ELEVATION - e.g. flat/effuse, raised, convex, dome-shaped/pulvinate, umbonate, umbilicate
MARGIN - edge of colony, e.g. entire, undulate, lobate, erose, filamentous, curled
COLOUR - by reflected light, e.g. white, cream, etc. || fluorescent, iridescent, opalescent, self-luminous || pigment - diffusible or non
SURFACE - e.g. glistening, dull, striated, granular, etc.
DENSITY - by transmitted light (ability to see through the colony) - opaque/transparent/translucent
CONSISTENCY - observed by picking up with loop, e.g. butyrous, viscid, friable, brittle, membranous
OTHER - odour, growth down into medium
NB: Characterisitcs are recorded under stated conditions of time, temperature & atmosphere, and are influenced by conditions of incubation.
AD. Describe how motility is achieved in some microbes, and how it may be visualised.
Give an example of how a type of bacterium might move in an aqueous environment.
- Motility is facilitated by hair-like appendages known as FLAGELLA; 10-20 um long and 0.2 um in diameter.
- They cannot be seen with LM w/o staining
- They originate in the plasma membrane
- Rotate, pushing against the surrounding medium, propelling the bacterium
Arrangements:
- Monotrichous - single
- Polar - single at one end
- Amphitrichous - at both ends
- Peritrichous - over entire surface of the cell
Staining: Mordants are used to precipitate on the flagella, making them thicker. The mordant is then stained & flagella can be seen under the LM.
E. coli (e.g.) moves in a straight line (run) when flagella rotate in synchrony. When the flagella rotate asynchronously, the cell begins to tumble. When the flagella rotate synchronously again, the cell changes direction.
AE. Aseptic techniques are methods of handling materials which:
A. involve isolating microbes only as pure cultures
B. minimise contamination of pure cultures and the environment
C. prevent epidemic outbreaks
D. ensure that cultures are never found to be contaminated
B. minimise contamination of pure cultures and the environment
AF. Which one of the following items is NOT an example of aseptic technique?
A. working carefully to ensure accuracy
B. working close to a Bunsen at all times
C. passing the neck of open containers through a flame at the beginning and end of
each manipulation
D. holding open containers in a near horizontal position
A. working carefully to ensure accuracy
AG. Jenny performed an oxidase test on a gram negative rod microbe as follows:
After sanitising the work space, with forceps she picked up a fresh oxidase test strip and placed on a clean glass slide. She used a sterile stick to transfer a large number of cells from a pure culture plate. This was sampled on the oxidase paper. The test was a partial false positive, as she soon found out the microbe to be E. coli, which should test negative for oxidase.
What small step regarding aseptic technique did Jenny forget to carry out?
Jenny should have flamed the forceps in the Bunsen, allowing them to cool, BEFORE she picked up the oxidase test strip. These forceps could have been contaminated with an oxidase positive organism such as Pseudomonas/Vibrio (both G-, rods).
Otherwise - her aseptic technique was relatively good and she didn’t use the nichrome wire, which can induce false positives due to zinc reacting with the substrate.
AH. What are the 2 main rationals behind aseptic technique?
- Ensure cultures remain free from contamination
2. Ensure the microbes you work with do not contaminate you or the environment
AI. In the Gram stain, after the primary stain, mordant and decolouriser have been added. Before the counterstain has been used, Gram positive organisms are stained ………………………….. & Gram negative are stained ……………………….. .
A. purple, purple
B. purple, colourless
C. purple, pink
D. pink, pink
B. purple, colourless
AJ. What is a mordant and how does it facilitate the Gram stain process?
A mordant sets dyes on a substrate material by forming a chemical coordination complex.
Iodine is referred to as a ‘mordant’ in Gram stains, but it is in fact a trapping agent. In the form of ‘iodide’ in Gram’s Iodine (Step 2), it binds to crystal violet (from the primary stain, Step 1) and traps it in the cell.
This will remain in G+ microorganisms after decolorization (with ethanol/acetone, Step 3), due to the multilayered nature of their peptidoglycan, but will be lost in G-‘s as the outer lipopolysaccharide membrane interacts with the alcohol decolorizer, leaving the inner peptidoglycan layer exposed, from which the crystal violet-iodine complexes wash out.
This is the basis of distinguishing between the gram morphologies.
AK. Phase contrast microscopy is valuable for observing:
A. viable, stained organisms
B. viable, unstained organisms
C. internal details of live microbes
D. the molecular structure of microbial cell components
B. viable, unstained organisms
AL. Suppose you were viewing through the microscope a Gram-stained field of uniform rods arranged in chains of varying lengths containing purple and some red
bacilli. You should conclude that:
A. the culture is an environmental isolate
B. you should repeat the stain as you have made a mistake
C. a mixed culture
D. some old cells are present in the chains
D. some old cells are present in the chains
AM. Fermentation of carbohydrate by bacterial cells producing energy involves all of the following EXCEPT:
A. aerobic breakdown of the carbohydrate
B. anaerobic breakdown of the carbohydrate
C. oxidation of the carbohydrate without an exogenous electron acceptor
D. the carbohydrate may act as both the primary electron donor as well as the final electron acceptor
A. aerobic breakdown of the carbohydrate
AN. OBLIGATE ANAEROBES can grow with/without oxygen BECAUSE:
they can metabolise energy aerobically/anaerobically. They gather mostly at the top of the tube* because aerobic respiration generates more ATP than either fermentation or anaerobic respiration.
Regarding the above,
a) The explanation correctly matches the subject.
b) The explanation inaccurately matches the subject.
c) The explanation is a true statement but the subject is incorrect.
d) Both the subject & explanation are false statements.
c) The explanation is a true statement but the subject is incorrect:
Obligate anaerobes CANNOT grow in oxygen, they are poisoned by it so they gather at the bottom of the tube where the concentration is the lowest.
FACULTATIVE ANAEROBES match the subject & description.
*NB thioglycollate broth
AO. Which Stage 1 Cowan & Steel Test utilises Kovac’s Reagent?
a) Catalase
b) Indole Production
c) Spore Stain
d) Oxidation-Fermentation
e) None of the above
e) none: the OXIDASE test uses Kovac’s reagent; it contains tetramethyl-p-phenylenediamine which serves as an alternative substrate for the oxidase enzyme. In the reduced state, the reagent is colourless; when oxidised becomes blue.
AP. Micrococcus luteus & Neisseria gonorrhoeae are both coccus, non-motile, catalase & oxidase positive and oxidative (aerobic).
What are two ways to differentiate these two microbes?
Morphology under light microscope should reveal:
- Gram-positive TETRAD formations of M. luteus
- Gram-negative DIPLOCOCCI formations of N. gonorrhoeae.
NB Neisseria species are typically in pairs but some types can appear in tetrad forms!
AQ. The physiological significance to a bacterium of production of catalase is:
A. to catalyse reactions involved in aerobic respiration
B. breakdown of the toxic end product of its own metabolism, hydrogen peroxide
C. breakdown of hydrogen peroxide, producing essential oxygen for cellular respiration
D. to support the cytochrome electron transport chain producing oxygen to act as the final electron acceptor
B. breakdown of the toxic end product of its own metabolism, hydrogen peroxide
AR. False positive catalase test results:
A. can occur when the test is performed on the open bench
B. can be obtained when some of the blood growth medium is picked up with the organisms being tested
C. can be obtained when testing a pathogen growing in liquid, broth blood culture
D. are never found, as the organism either produces catalase enzyme or it does not
B. can be obtained when some of the blood growth medium is picked up with the organisms being tested
AS. False negative catalase test results:
A. can occur when the culture growth being tested is dead
B. can be obtained when the culture being tested is in exponential growth phase
C. can be obtained when the hydrogen peroxide reagent used is old and has degraded to water
D. are never found, as the organism either produces catalase enzyme or it does not
C. can be obtained when the hydrogen peroxide reagent used is old and has degraded to water
AT. Regarding the Catalase Test,
a) Mixing a sampled culture into the hydrogen peroxide sample will increase visible bubbles
b) Aerobic or Anaerobic bacteria can produce catalase.
c) It tests for cytochrome C catalase in the electron transport chain of certain bacteria.
d) Catalase catalyses the breakdown of a metabolic end product which might otherwise accumulate and kill the organism which produces it.
e) a & d
CORRECT: d)
a) Sample should be PLACED, NOT mixed as this will REDUCE bubble visibility and the confirmation of oxygen gas. (immerse! not emulsify).
b) Anaerobic bacteria do NOT produce catalase (especially obligate anaerobes; poisoned by oxygen). However some facultative anaerobes do.
c) This refers to (cytochrome C) Oxidase
