GRAM STAINING power point presentation ppt

GRAM STAINING
PRESENTER:DR.A.SHAMILI
MODERATOR:DR.VENKATESH V.N

OBJECTIVE……..
Introduction
History
Principles of gram stain (GS)
Modification of GS
Procedure of GS
Interpretation of various specimens
Applications
Limitations
QC

The structure details of the cells are not seen under light
microscope due to lack of contrast
It is necessary to increase the contrast and there by increase the
visibility

It is the most common differential staining method used in
Microbiology
It differentiates bacteria into 2 groups
GRAM POSITIVE
GRAM NEGATIVE

FILM MAKING AND FIXATION
One loopful of sample is taken up with inoculating loop and spread
it thinly on the slide.

Note: For solid materials (Cultures on agar)
(i) It is necessary to place a loopful of normal saline on the slide.
(ii) Loop is then sterilized, minute quantity of material obtained
by just touching the growth, is transferred to the drop throughly
emulsified and eventually spread on the slide.

TYPES OF FIXATION
Why Fixation of smear is important?
Fixation helps to protect the internal structures of cell in fixed
position.
HEAT FIXATION
Gentle flame heating an air dried film.
(BACTERIAL SMEARS)
METHANOL FIXATION
Used for blood smears.

Marking diamond or grease pencil may be used to write on the
flim bearing side of the slide.
Stick on paper labels should not be used due to may be washed
off during the staining procedure.

HISTORY
 Devised by Danish Physicist – HANS CHRISTIAN JOACHIM
GRAM
 In 1883
 Published his work in 1884
 Studied on lung tissues of patients who had died of pneumonia–
distinguished Klebsiella from Streptococcus pneumoniae.

PRINCIPLE/THEORIES OF
GRAM STAINING

pH theory(acid protoplasm theory)
• Protoplasm: There is a more acidic protoplasm (pH 2–3) in the
gram-positive cells, which is responsible for retaining the basic
dye more strongly than the Gram-negative bacteria.

Different kinds of bacteria react differently to the Gram stain,
because structural differences in their cell walls affect the retention
or escape of a combination of crystal violet and iodine, called the
crystal violet-iodine (CV-I) complex.

• CELL WALL THEORY:
• Iodine makes the protoplasm more acidic and serves as mordant,
i.e. iodine combines with dye to form a dye-iodine complex and
fixes the dye in bacterial cell.
• When applied to both gram-positive and gram-negative cells,
crystal violet and then iodine readily enter the cells. Inside the
cells, the crystal violet and iodine combine to form crystal violet-
iodine (CV-I) complex

GRAM POSITIVE CELL
The complex crystal violet-iodine (CV-I) complex is larger than the
crystal violet molecule that entered the cells, and because of its
size, it cannot be washed out of the intact peptidoglycan layer of
gram-positive cells by acetone/alcohol. Consequently, gram
positive cells retain the color of the crystal violet

GRAM NEGATIVE CELL
• They are thinner, have a smaller amount of peptidoglycan, less extensively
cross-linked and contain a high percentage of lipids.
• There is a layer of lipopolysaccharide as part of their cell wall.
• They dissolve during treatment with acetone alcohol, forming larger pores
in the cell wall, and the CV-I complex is washed out through the thin layer
of peptidoglycan causing out-flow of dye-iodine complex and take up
counter stain, thus appearing pink (gram negative).

Magnesium–ribonucleate theory
 It has been found that Gram positivity depends on the integrity of cell
wall and presence of specific magnesium–ribonucleate–protein complex.
 The gram-positive bacteria become gram-negative when cell wall is
damaged.
 Thus, gram-positive bacteria may become gram-negative, if the cell is
aging, mechanically ruptured or by the action of ribonuclease

GRAM STAINING power point presentation ppt

LIPID CONTENT THEORY
(STERN THEORY)
More recent one
GP contains 4 % lipids in their cell wall
 GN contains 20 % lipids in their cell wall
Lipids are easily washed by decolourizer in GN

IOSELECTRIC POINT THEORY
Isoelectric point is the pH where ionization of amphoteric
compounds is at maximun
Isoelectric point of GP is pH - 2 to 3
Isoelectric point of GN is pH - 4 to 5
There are more unsaturated acids in GP are oxidized during iodine
treatment and more acidity is produced in cytoplasm
Thus GP able to retain the primary stain

JENSEN’S MODIFICATION(Gonococci & Meningococci):This
method involves use of Absolute alcohol as decoloriser and Neutral
red as counter stain.
Preston and morrels gram method: Used Crystal violet as
primary stain & iodine acetone as decoloriser which gives good
results and strengthens gram positive staining.
BROWN and BRENN modification : used for actinomycetes.

Weigert Modification: Uses gentian violet as primary stain and
Aniline xylol as decoloriser which is useful for tissue sections.
Kopeloff and Beermans modification: uses Methyl violet as
primary stain and acetone or acetone alcohol, useful for the
identification of anaerobes and in bacterial vaginosis.

Specimens
Clinical specimens, generally excluding throat swabs, nasal swabs,
sputum from cystic fibrosis patients, fecal material, and prosthetic
devices.
Direct smears are particularly useful for wounds, eye lesions, body
tissues, and certain discharges.

Rejection criteria
1. Gram stains are of little value as direct smears of stool, blood, or
throat. They are not helpful for sputum from cystic fibrosis patients.
2. Stains are not part of standard protocols for evaluation of
catheter tip specimens

Reagents
 Crystal violet a. Hucker’s modification b. Kopeloff’s crystal violet
 Iodine a. Gram’s reagent b. Kopeloff’s iodine
 Decolorizers
a. Slowest: ethanol, 95%
b. Intermediate: acetone-alcohol (1) Mix 50:50. (2) Combine in brown glass
bottle, label with 1-year expiration date, and store at room temperature.
c. Fastest: acetone.
 Counterstain
a. Safranin b. Carbol fuchsin c. Basic fuchsin (0.8, 0.1, or 0.2%)

REAGENTS
A
CRYSTAL VIOLET OR METHYL VIOLET
ABSOLUTE ALCOHOL (100% ETHANOL)
DISTILLED WATER
10g
100ml
1 litre
B
GRAMS IODINE:
IODINE CRYSTAL
POTTASSIUM IODIDE
DISTILLED WATER
10g
20g
1000ml
C
DECOLOURIZER
ACETONE OR ABSOLUTE ALCOHOL OR ACETONE ALCOHOL OR IODINE ACETONE
D
COUNTER STAIN
DILUTE CARBOL FUSCHSIN
DILUTED WITH 10-20 TIMES ITS VOLUME OF WATER

PROCEDURE:
Four steps in grams stain procedure (After fixation)

QUICK GRAM METHOD FOR
SINGLE SLIDES
 When the staining time is very short and a single slide has to be stained
this method yields fairly good results
 Flood the slide with crystal violet allow to act for 5ses
 Tip off the stain ,flood the tilted slide with iodine for 5secs
 Tip off iodine and flood with acetone for 2 secs, wash with water
 Add basic fuschin for 5secs
 Wash, blot dry and observe

QUICK GRAM METHOD FOR
MULTIPLE SLIDES
Difficult procedure
Slides are placed horizontally on the rack, provided that each
reagent completely washed off with water and each succeeding
reagent is added to replace residual water.
Needs slowly acting decolorizer like iodine acetone.

INTERPRETATION OF GRAM
STAINING RESULTS
Screening under low power field
Screening under oil immersion field

Screening under low power field
Observe for stain crystals
Determine if the smear is properly decolorised
Determine if the smear thickness is appropriate
Representative fields
Host cells

Semiquantification of
neutrophils and organism in GS

Screening under oil immersion field
Fields to examine (20-40 fields good quality OIF)
Gram variability
Artifacts

GS FROM BLOOD CULTURE
BROTH
Performed on positively flagged blood culture broth
Utility for clinical team: Helps initiating or modifying the
treatment
Utility for microbiology team: to decide on further workup of the
culture

1.GS OF CSF/BODY FLUIDS
SAMPLE
Centrifugation
Sediment------- GS
Supernatant------- antigen detection / molecular assays
Cytospin centrifuge
Centrifuge the specimen
Preparation of smear

Uncentrifuged specimen
Smear preparation (HEAPED DROP METHOD)
Screening
Minimum 20-40 fields should be screened

2. GS OF URINE SPECIMEN
Less sensitive
Laborious
Does not offer additional effects
Uncentrifuged specimen
10 fields should be screened
Presence of at least one organism indicates significant bacteriuria

3.GS OF RESPIRATORY
SPECIMEN
Assessment the specimen quality
Bartletts grading system
Murray-Washington criteria
Indication where grading is not used
Reporting of organism in RS specimen

Assessment the specimen quality

SPECIMEN REJECTED :
Final score of 0 or less -------
Rejected
Note: Specimen is
unsatisfactory for culture;
repeat sputum specimen can be
requested if clinically indicated.
SPECIMEN ACCEPTED :
A final score of ≥1------
accepted

Indications where Grading is
not required
Not used to assess the LRT specimens like BAL, Protected
specimen brushings, lung aspirate
Induced sputum
Neutropenic patient
Atypical agents causing pneumonia
Foreign device
Cystic fibrosis

Reporting of GS in RS
specimen
COMMENSAL FLORA
GPB
GPC IN CHAINS
COLONIZER FLORA:
Pneumococci
Haemophilus influenza
Moraxella
GNB

The clinical microbiologist should never report the morphological
appearance of normal flora individually in sputum GS report as it
may mislead the clinician to interpret these organism as potential
pathogens

Reporting of potential
pathogens
Morphotypes of organism can be reported if they are suspected to
be pathogenic and differentiated from colonizer

GS of ocular specimen
The structure seen in gram stained smear in ocular specimens are
neutrophils, bacteria, yeast, hyphae, Acanthamoebae, microsporidia,
Nocardia

GS OF EXUDATE SPECIMEN
Atleast 20-40 cellular field of the smear are examined
Pus cells and organism are graded from 1+ to 4+

Gram positive cocci in
cluster
Gram positive budding yeast
cells

Streptococcus pneumoniae
lancet-shaped diplococci
in Gram stain,
Gram-negative intracellular
diplococci,
which are diagnostic for
gonorrhea in urethral discharge

Vibrio cholerae (Gram stain):
Curved comma-shaped gram-
negative rods (fish in stream
appearance).
Gram stained smear of feces -
curved gram negative bacilli,
comma or S-shaped or spiral (gull
wing) shaped

LIMITATIONS OF GRAMS
STAINING
Thick smear– gram negative organism appears as gram positive--
Integrity of cell wall disrupted.
GP anaerobes easily decolourise.
Over-decolourisation with acetone.

Gram stain-positive, culture-negative specimens may be the result
of contamination of reagents and other supplies, presence of
antimicrobial agents, or failure of organisms to grow under usual
culture conditions (medium,atmosphere,etc.).
 False Gram stain results may be related to inadequately collected
specimens or delays in transit.

APPLICATIONS
Gram positive & Gram negative differentiation
Idea for corresponding biochemical tests
To start empirical Rx
For fastidious organisms (Haemophilus takes time)
Anaerobic organisms
Yeast (Candida and Cryptococcus)
Quality of specimen (More pus cells and less epithelial cells)

QUALITY ASSURANCE OF
DIRECT BC GRAM STAIN
 This is one of the key performance indicator in microbiology
 Accuracy should be more than 90%

QUALITY CONTROL
 A. Check appearance of reagents daily.
 If crystal violet has precipitate or crystal sediment, refilter before use.
 Limit reuse of working stain containers by discarding at least monthly.
 B. Test laboratory staining procedure prior to use of new lots of each staining and
decolorizing reagent and at least weekly thereafter, using a gram-positive and
gram-negative microorganism.
 C. Take corrective action when stained smear preparations show evidence of poor
quality, stains are difficult to interpret, or interpretations are inaccurate.

GRAM STAINING power point presentation ppt

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