Staining

Question 1

what happens when you gram stain Candida albicans?

Answer 1

•   C. albicans retains crystal violet and appears “Gram-positive”
•   However, C. albicans is NOT a Gram-positive organism (yeast!!)
•   cells are significantly larger than bacteria
•   cells grow ‘pseudohyphae’ when grown in culture containing serum

Question 2

Why is the zhiel neeson method used for staining?

Answer 2

•   some bacteria cannot be stained by the Gram method
•   Mycobacteria have a wax-like coat made of mycolic acid
•   Mycobacteria have a Gram-positive cell wall morphology

carbolfuchsin, heat -> ethanol, hydrochloric acid
(destain) -> methylene blue (counter stain)
acid fast cells are red

Question 3

How does the Lactophenol Cotton Blue Stain work?

Answer 3

•  lactophenol serves as a mounting fluid
•  phenol kills the mould and prevents cell lysis
•  cotton Blue stains the chitin in the cell wall
•  slide shows hypha (1), fruiting body (2) and spores (3)
example = penicillium

Question 4

coagulase test

Answer 4

•  Identification of Staphylococcus aureus
•  Cell surface-bound protein that mediates fibrin polymerisation
S. aureus has coagulase on its cell surface and this enzyme mediates fibrin polymerization that can be seen as clot formation in plasma

Question 5

Oxidase test

Answer 5

• Some obligate aerobic bacteria produce cytochrome C oxidase as part of the respiratory chain.
  -  this enzyme mediates transfer of e- to O2 (respiratory chain)
• incubation of bacteria with oxidase reagent (N,N,N’,N’ - tetramethyl-p-phynylenediamine dihydrochloride results in a dark blue stain.
  Oxidase-positive bacteria:
• Pseudomonas

Question 6

Catalase test

Answer 6

Used to differentiate between STaphylococci and Streptococci.

Question 7

Microbact strips

Answer 7

-  miniaturised test systems that uses standard biochemical identifications
-  each position on the strip is an independent test reaction
-  test is for specific enzyme, specific substrate or specific metabolic product
-  added indicator to monitor biochemical reaction
Reactions are assessed by change of color, either by the reaction product itself or by a ph indicator. There is a different Microbact strips available, e.g. the Microbact 12E test is used to identify species of the family Enterobacteriaceae

Question 8

what is selective agar?

Answer 8

contains inhibitors to prevent growth of certain organisms

Question 9

What is differential agar?

Answer 9

contains indicators to differentiate organisms

Question 10

is Sabouraud agar selective of differential?

Answer 10

Selective, - selective for fungi, non-differential

- low pH suppresses growth of most bacteria

Question 11

Is Eosin-methylen blue agar (EMB) selective of differential?

Answer 11

• selective for Gram-negative bacteria
• aniline dyes are toxic for Gram-positive bacteria
• differentiates lactose fermenters: pink=weak/moderate
  green=strong/rapid

Question 12

Is MacConkey agar selective of differential?

Answer 12

• selective for intestinal pathogens
• bile salt inhibits non-enteric bacteria
• differentiates lactose fermenters (pink)

Question 13

is Blood agar selective or differential?

Answer 13

• growth of many fastidious bacteria

- differentiates for hemolytic reactions

Question 14

is Mannitol Salt agar selective or differential?

Answer 14

• selective for haloduric bacteria (Staphylococci)
• differentiates mannitol fermenters (yellow)
• S. aureus is the only Staph that ferments mannitol

Question 15

is Bile-Esculine agar selective or differential?

Answer 15

• selective for enteric bacteria
• oxgall inhibits non-enteric bacteria
• esculine hydrolysis gives a dark brown color

Question 16

Antibiotic susceptibility test describe it

Answer 16

Antibiotics can be used to diffferentiate between certain species e.g. S. pyogenes is sensitive to bacitracin, but streptococcus dysgalactiae is resistant. Tests are conducted on lawn agar plates with antibiotic discs to see if they are sensitive, intermediate or resistant. You decide based on how much there is of a lack of growth.

Question 17

What are the different methods based on antibody-antigen interaction?

Answer 17

Cell agglutination, latex bead agglutination, western blot, immunofluorescence microscopy

Question 18

Cell agglutination

Answer 18

Antibodies recognise component on cell surface resulting on visible clumping. Bassis of serotyping where serum is used to distinguish between different types of a given species (serotypes) based onspecefic antibodies to a variable surface molecule i.e. E.coli O157 H7

Question 19

Latex bead agglutination

Answer 19

Antibodies can be bound to latex beads. A positive reaction leads to visible clumping of the latex beads. Used for e.g. Lancefield group typing of Streptococci.

Question 20

Western blot

Answer 20

Used for identification of proteins or antibodies. Proteins are separated on a gel according to their molecular weight and then immobilized by blotting onto a nitrocellulose membrane. Antibodies are added to the membrane and specific binding to protein occurs. A second antibody labelled with an enzyme e.g. alkaline phosphatase, Ap, and with specificity to the Fc region of the first (antigen-specific) antibody is added. Binding is monitored by adding a substrate that is converted by AP resulting in detectable protein bands.

Question 21

Immunofluorescence microscopy

Answer 21

same principle as in Western Blot. The secondary antibody is labelled with a fluorophore (e.g. FITC), cyanine or rhodamine which can be detected using a fluorescence microscope

Question 22

What are the two types of DNA based assays?

Answer 22

Restriction fragment length polymorphism (RFLP) (DNA　ｆｉｎｇｅｒｐｒｉｎｔｉｎｇ。　
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Question 23

RFLP

Answer 23

small mutation on the genome can lead to destruction or generation of a recognition sequence for a restriction enzyme. This leads to changes in the length of DNA fragments after treatment with DNAse and can be detected after separating the DNA fragments on a gel. Mainly used for epidemiological studies.

Question 24

PCR

Answer 24

uses specific oligo DNA primers and a heat-stable DNA-polymerase to amplify very small amounts of DNA. Important method to identify organisms that cannot easily be grown in culture. Also important in forensic science.