Gram staining is a universal procedure uniquely used in Bacteriology, Microbiology. This is mainly used in case of bacteria to separate them into two categories as Gram Positive & Gram Negative. This technique uses chemical like crystal violet and Safranin .

1. GRAMSTAINING&other staining
procedures
Represented by WASIM AKRAM
BSc Nursing
ADWIKA INSTITUTE OF NURSING

2. GRAMSTAINING
 GRAM STAINING IS A METHOD OF DIFFERENTIAL
STAINING WHICH DIFFERENCIATES THE BACTERIA
INTO TWO LARGE GROUPS :
 THE ONE GROUP IS CALLED AS GRAM-POSITIVE
ORGANISMS AND THEY RETAIN PRIMARY DYE , i.e.
CRYSTAL VIOLET .
 ANOTHER GROUP IS CALLED AS GRAM-NEGATIVE
ORGANISMS AND THEY TAKE UP THE
COUNTERSTAIN, i.e. SAFRANIN .
 THIS METHOD IS MOST WIDELY USED IN
MICROBIOLOGY BACTERIOLOGY ) .

3. PRINCIPLE
 IN 1884 , CHRISTIAN GRAM INTRODUCED THIS
TEST AND HUCKER MODIFIED IT IN 1921 .
 THE BASIC PRINCIPLE OF GRAM-STAINING IS
BASED ON THE DIFFERENCE IN THE CELL WALL
COMPOSITION OF DIFFERENT BACTERIA .
 THE BACTERIA RETAINING CRYSTAL VIOLET
COLOR IN THEIR CELL WALL DUE TO HIGH
PEPTIDOGLYCAN CONTENT WHEARAS SOME
BACTERIA DO NOT RETAIN IT DUE TO LOW LIPID
CONTENT AND TAKE SAFRANIN AS
COUNTERSTAIN .
 THE ONE THAT TAKES PRIMARY STAIN APPEARS
PURPLE IN COLOUR – GRAM POSITIVE AND
OTHERS WHICH TAKE COUNTERSTAIN ARE
GRAM NEGATIVE ORGANISMS .

4. OBJECTIVES/
USES
 IT HELPS IN DIFFERENTIATION BETWEEN GRAM-
POSITIVE AND GRAM-NEGATIVE BACTERIA .
 IT HELPS TO OBSERVE THE MORPHOLOGY AND
ARRANGEMENT OF CELLS .
 IT IS USEFUL IN THE PRELIMINARY DETECTION AND
IDENTIFICATION OF ORGANISMS FROM CLINICAL
SPECIMENS LIKE CEREBROSPINAL FLUID IN CASE
OF MENINGITIS WHERE TREATMENT HAS TO BE
STARTED AT THE EARLIEST .
 IT HELPS IN THE SELECTION OF CULTURE MEDIA
FOR GROWING ORGANISMS IN LABORATORY .
 IT IS USEFUL IN THE SELECTION OF ANTIBIOTICS
FOR TREATMENTS .

5. SPECIMENFOR
STAINING
 THE GRAM STAINING CAN BE PERFORMED ON
THE SMEARS PREPARED FROM CLINICAL
SAMPLES , TISSUES , BODY FLUIDS AND BROTH
CULTURES OR THE COLONIES PICKED FROM
CULTURE PLATES .
 MOSTLY YOUNG CULTURES ARE PREFERRED
BECAUSE THE ORGANISM IN THE OLD
CULTURES TENDS TO LOSE GRAM-POSITIVE
CHARACTER .

6. PROCEDURE:
1.SMEAR
PREPERATION
 IT IS PREPARED BY TAKING A DROP OF NORMAL
SALINE ON A CLEAN SLIDE .
 A COLONY FROM THE SOLID MEDIA PLATE IS
PICKED WITH THE HELP OF INOCULATING WIRE
LOOP AND IS EMULSIFIED IN NORMAL SALINE .
 NOW AFTER AIR DRYING , THE SMEAR IS HEAT
FIXED AND IS THEN SUBJECTED TO STAINING
PROCEDURE .

7. 2.STEPSOF
GRAMSTAINING
PROCEDURE
1. FLOOD THE FIXED SMEAR WITH THE CRYSTAL VIOLET
SOLUTION AND LEAVE IT FOR ONE MINUTE .
2. AFTER POURING OUT CRYSTAL VIOLET , RINSE IN
RUNNING WATER GENTLY , SO THAT THE DYE IS NOT
WASHED OFF TOTALLY .
3. POUR IODINE SOLUTION ON THE SMEAR AND LEAVE
IT FOR ONE MINUTE.
4. RINSE GENTLY IN WATER .
5. DECOLORIZATION IS DONE WITH ALCOHOL BY
KEEPING IT ON THE SMEAR FOR 30 SECONDS .
6. AGAIN RINSE WITH FLOWING WATER .
7. SAFRANIN IS USED AS COUNTERSTAIN AND IS KEPT
ON THE SMEAR FOR 30 SECONDS .
8. AGAIN RINSE THE SLIDE IN RUNNING WATER AFTER
REMOVING EXCESS OF COUNTERSTAIN .
9. AIR DRY THE SLIDE AND EXAMINE UNDER THE OIL
IMMERSION OBJECTIVE OF A MICROSCOPE .

8. 3.
INTERPRETATION
OFRESULTS
 THE GRAM POSITIVE BACTERIA LOOK PURPLE
IN COLOUR AND GRAM NEGATIVE BACTERIA
LOOK PINK IN COLOUR .

9. 4.LIMITATIONS
OFTHE
PROCEDURE
 THE GRAM STAINING IS USEFUL IN
CHARACTERIZING BACTERIA , YET IT IS NOT
APPLICABLE TO OTHER GROUPS OF MICRO-
ORGANISMS LIKE PROTOZOA AND FUNGI .
 YEASTS , HOWEVER GET STAINED AS GRAM
POSITIVE ORGANISMS .
 ALTHOUGH , ACCURATE RESULTS CAN BE
OBTAINED AFTER EXPERIENCE .
 THE CONTAMINATION OF THE CLINICAL
SAMPLES MAY GIVE RISE TO FALSE GRAM
REACTIONS .

10. 5.ROLEOF
EACHSTEPS
 CRYSTAL VIOLET IS THE PRIMARY STAIN AND
STAINS ALL BACTERIA PURPLE OR VIOLET .
 GRAM’S IODINE IS A MORDANT WHICH
COMPLEXES WITH CRYSTAL VIOLET AND FIXES
THE DYE TO THE BACTERIA .
 ACETONE OR ALCOHOL IS A DECOLORIZING
AGENT WHICH CAUSES THE DYE-IODINE
COMPLEX TO LEAK OUT IN GRAM NEGATIVE
BACTERIA .
 DILUTE CARBOL FUCHSIN IS A COUNTERSTAIN
WHICH STAINS GRAM NEGATIVE BACTERIA PINK
.

11. THEORIESOF
GRAMSTAINING
1. CELL WALL PERMEABILITY / LIPID CONTENT
THEORY
2. ACIDIC PROTOPLASM THEORY
3. MAGNESIUM RIBONUCLEATE COMPLEX
THEORY .

12. ACID-FAST
STAINING
 THIS IS ANOTHER METHOD FOR DIFFERENTIATING
ACID FAST FROM NON-ACID FAST BACTERIA .
 THIS METHOD WAS INTRODUCED BY PAUL EHRLICH
IN 1882 BUT MODIFIED BY ZIEHL NEELSEN . NOW IT
IS ALSO KNOWN AS ZIEHL NEELSEN STAINING
METHOD.
 THIS METHOD IS VERY USEFUL IN STAINING OF
 MYCOBACTERIUM TUBERCULOSIS
 MYCOBACTERIUM LEPRAE .
 THESE BACTERIA DO NOT TAKE THE SIMPLE STAINS
DUE TO THE PRESENCE OF VARIETY OF FATTY ACIDS
; i.e. MYCOLIC ACID IN THEIR CELL WALL . BUT
WHEN STAINED WITH HOT CONCENTRATED CARBOL
FUCHSIN , THE ORGANISMS STAIN BRIGHT RED .

13. BILESTAINING
 BILE STAINING DEPENDS ON THE LIPID
CONTENT.
 SOME EGGS ALSO HAVE RECEPTORS FOR BILE
ON THEIR SURFACE ( SHELL ) .
 SOME BILE STAINED OVA / EGGS ARE OF :
 ASCARIS LUMBRICOIDES
 FASCIOLA GIGANTICA
 TRICHURIS TRICHURA
 TAENIA SAGINATA
 TAENIA SOLIUM

14. CONCLUSION
NOW A DAYS STAINING PROCEDURES ARE VERY
COMMON TO DETECT THE BACTERIAL CATEGORY . BY
DOING THIS STAINING PROCESS EASILY WE COME TO
KNOW THE WAY BY WHICH THE BACTERIA CAN HARM
HUMAN BODY . THERE ARE SOME STAINING
TECHNIQUES LIKE GRAM STAINING , ACID-FAST
STAINING AND BILE STAINING . IN THESE , GRAM
STAINING IS WELL FAMOUS AND USEFUL WORLDWIDE
.

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