Flashcards in Staining Deck (24)
what happens when you gram stain Candida albicans?
- 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
Why is the zhiel neeson method used for staining?
- 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
How does the Lactophenol Cotton Blue Stain work?
• 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
• 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
- 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.
Used to differentiate between STaphylococci and Streptococci.
- 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
what is selective agar?
contains inhibitors to prevent growth of certain organisms
What is differential agar?
contains indicators to differentiate organisms
is Sabouraud agar selective of differential?
Selective, - selective for fungi, non-differential
- low pH suppresses growth of most bacteria
Is Eosin-methylen blue agar (EMB) selective of differential?
- selective for Gram-negative bacteria
- aniline dyes are toxic for Gram-positive bacteria
- differentiates lactose fermenters: pink=weak/moderate
Is MacConkey agar selective of differential?
- selective for intestinal pathogens
- bile salt inhibits non-enteric bacteria
- differentiates lactose fermenters (pink)
is Blood agar selective or differential?
- growth of many fastidious bacteria
- differentiates for hemolytic reactions
is Mannitol Salt agar selective or differential?
- selective for haloduric bacteria (Staphylococci)
- differentiates mannitol fermenters (yellow)
- S. aureus is the only Staph that ferments mannitol
is Bile-Esculine agar selective or differential?
- selective for enteric bacteria
- oxgall inhibits non-enteric bacteria
- esculine hydrolysis gives a dark brown color
Antibiotic susceptibility test describe it
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.
What are the different methods based on antibody-antigen interaction?
Cell agglutination, latex bead agglutination, western blot, immunofluorescence microscopy
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
Latex bead agglutination
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.
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.
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
What are the two types of DNA based assays?
Restriction fragment length polymorphism (RFLP) (DNA ｆｉｎｇｅｒｐｒｉｎｔｉｎｇ。
Ｐｏｌｙｍｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ
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.