Lecture 4 for 122 Flashcards
Application of Gram stain in Clinical lab
Usually the first step in the micro lab.analysis of a clinical material from an infected body site is a direct microscopic examination by preparing a smear and doing a gram stain.
This helps the technologist to determine if there are gram positive or negative bacteria in the specimen
They can also determine the ,
cellular morphology and arrangement ,which may provide clues to the organism’s identity.
In some clinical situations Gram Stain is a STAT.e.g CSF.
The effects of improper gram stain on patient results
On A well done gram stain smear , gram positive cells would look purple and gram negative cells would look pink, however if not done properly, then that affects the patient results
Factors resulting in improperly done gram staining.
Improper density of the smear: The quality of the smear prepared is very important.
The smear has to be of proper density. If the Cell density of the smear is extremely thick ,then the smear may not decolorize as rapidly as one of ordinary density. Causing over or under decolorized smears.
Factors resulting in improperly done gram staining.
Concentration and freshness of the Gram stain reagents:
If the stains are old then precipitates may be present ,when stained with such stains some flecks of precipitated stains may be present causing artifacts on the slide, which may cause difficulty in interpretation of results.
Factors resulting in improperly done gram staining
Length and thoroughness of washing after crystal violet, and the amount of water remaining on the slide when iodine is applied.
- Age of bacterial cultures. Gram stain reactions are reliable only for cultures up to 24 hours old. Variability of Gram stain reactions in old cultures is often related to cell wall integrity and permeability.
- Over or under decolorizing of the smear by decolourizer.
The most important step of the Gram stain procedure is the decolorization step,
which is based on the ease with which the crystal violet-iodine complex is released from the cell on the application of the decolorizer.
Over-decolorizing will result in the loss of the primary stain, causing Gram positive bacteria to appear Gram negative..
Under decolourization
Under-decolorizing, however, will not completely remove the crystal violet-iodine complex, causing
Gram negative bacteria to appear Gram positive
Effects of improper staining
A slide is not appropriate for examination if the host cells are stained blue instead of red, indicating that the smear was under-decolorized.
A slide is also not acceptable for examination if microorganisms that should be gram-positive appear pink. This may indicate that either the Gram’s iodine was not applied or the slide was over-decolorized.
As such slides are unacceptable , that would affect the patient results, hence very important to do the procedure properly to avoid such problems
Action to be taken if slide is unacceptable
Always notify the instructor/supervisor if you find the slide is nor proper.
If enough sample is available, prepare a new smear and stain the slide properly.
If it is impossible to prepare a new smear, then remove immersion oil from the smear using xylol.(use proper PPE when handling Xylol)
If the smear is under-decolorized, repeat the decolorization and counterstain steps.
If the smear is over-decolorized, the slide should be stained again
To make sure the slides stained are proper ,it is important to run controls.
Staining gram-positive and gram-negative control slides along with the patient’s smear would confirm that proper staining technique was used.
Acid-Fast Stain
useful in the identification of acid-fast bacteria(AFB)
Cell walls of some acid-fast bacteria such as Mycobacterium, M.leprae, , M.tuberculosis
consists of as much as 60% of mycolic acid, whereas the rest is peptidoglycan. rapid and preliminary diagnosis of tuberculosis.
AFB stain is also known as the Ziehl-Neelsen acid-fast stain(ZN method).
sputum sample or a CSF
Mycolic acid is a waxy substance that gives the acid-fast cells a
higher affinity for the primary stain
resistance to decolorization by acid-alcohol solution.
A good sputum sample is one with more of
thick mucus or phlegm that is expelled from the lower respiratory tract (bronchiand lungs) through coughing;
it is not saliva or spit.
Even when seen microscopically it should have < 25 squamous epithelial cells.
cold staining
Kinyoun modification
Steps for Acid fast stain
Done in a beaker over hot plate
Primary stain - Carbol Fuchsin. Stain is alcohol based
Heat slide slide-Heat enhances the entry of carbolfuchsin into cells.
3.Decolorize with 3% HCL- alcohol acid-alcohol-Carbolfuchsin is more soluble in cellwall lipids than acid alcohol,so resists the decolorization .Non acid fast bacteria the cell wall lacks the lipid component and so carbolfuchsin is rapidly removed.
4.Counterstain with methlene blue.
So acidfast bacteria are-pink s
Non acid fat bacteria/background cells - blue
Kinyoun modification
Carbolfuchsin stain:Basic fuchsin, 0.3 gEthanol, 95% (vol/vol)- 10 mlPhenol, heat-melted crystals- 5 mlDistilled water, 95 mlDissolve the basic fuchsin in the ethanol; then add the phenol dissolved in the water.
Mix and let stand for several days. Filter before use.
Decolorizing solvent: 3% acid-alcohol solution
Ethanol, 95% (vol/vol) -97 ml
Hydrochloric acid (concentrated), 3 ml
Counterstain: Methylene blue chloride, 0.3 g
How much normal flora
A normal human has approximately 10^13 body cells and 10^14 individual
Normal flora are found on
the skin,eyes,nose,mouth,in the upper thorat,lower urethra, intestine,stomach
However there are certain body sites that are sterile:
Blood - it is a connective tissue not a fluid
Body fluids
Tissues
Biopsies Bone marrow
