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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.
4. 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.
5. 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 (bronchi and 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 g Ethanol, 95% (vol/vol)- 10 ml Phenol, heat-melted crystals- 5 ml Distilled water, 95 ml        Dissolve 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
Biopsies Bone marrow


Body Fluids include:

Cerebral Spinal fluid(CSF)
Pleural fluid(from the lungs)
Synovial fluid(from the joints)
Pericardial fluid( from around the heart)
Peritoneal fluid(from around the abdomen)
Amniotic fluid
Urine when its in the bladder once it passes through a urethra
Any culture positive from these samples indicate a disease


Non-sterile body sites are



GI specimen:

Most of the bacteria that cause diarrheal disease belong to the large family of G-ve bacteria called the Enterobacteriaceae .
Enterobacteriaceae are gram negative rods
They include some normal flora and some pathogens.
They are commonly referred to as enterics
Most labs routinely culture fecal specimens for Salmonella, Shigella, Campylobacter and E.coli, particularly E.coli 0157:H7

We don’t do gram stain because GI bacteria are all Gram neg so nothing substantial will be picked up


Vaginal Specimens:

The vaginal specimen is collected during a pelvic examination.
The specimen is collected using a sterile polyester tipped swab on a plastic shaft since,
Cotton has certain toxic ingredients which has proved toxic to N.gonorrhoeae and the wooden shaft is toxic to Chlamidya trachomatis


Specimen Collection

the right sample,
collected at the right time,
transported in the right way to the right laboratory


Collection guidelines must emphasize two important aspects:

1.Collection of the specimen before the administration of antimicrobial agents
2.Prevention of contamination of the specimen with normal flora of the body


Sample Collection and Transportation:

All microbiological specimens must be collected with the correct type of swab or in sterile screw cap containers, which are leak-proof
In almost all microbiological work sterile polyester or rayon swabs are used, since cotton swabs contain ingredients which may inhibit some morgs.
If the specimen is to be transported to another lab than the correct type of transport medium should be used.

Sterile containers
Amie’s or Stuart’s transport media
Formalin for Stool samples for parasites
Blood Culture Bottles
Syringes (no needle) for sterile biopsy fluids
Anaerobic transport media


Transport medium

1. Sterile
2. Maintains the viability of microorganisms
3. Non-nutritive


Amie’s Transport Media

Small amount of agar
Protects from drying
Minimal media (no nutrients)
Buffered to minimize fluctuations in pH
Charcoal may be added to absorb toxins enhance survival of fastidious organisms (e.g. Neisseria gonorrhoeae) recommended for throat, vaginal, and wound samples


In the formulation of a transport medium, Charcoal

neutralizes fatty acids that are toxic to microorganisms.


The Chloride salts in transport medium supply

essential electrolytes for transport and osmotic balance


Phosphates in transport medium act as

a buffer system


Sodium thioglycollate in transport medium

suppresses oxidative changes and provides an anaerobic environment.