Staining

By
Dr Sarfaraz Hussain Tunio
Rigid Thick walled Bacteria
Free Living (Extra cellular)
Gram positive

Bacilli

Gram Negative
These all Bacteria can not be stained with Grams staining

ZN Staining Mycobacterium
Acid fast Bacteria (AFB) Chlamydia
Giemsa Staining
Non free living (obligate intracellular) Ricketsia
Thin walled walled Bacteria (Flexible) Borrelia
( spirochetes) Warthin starry stain silver impregnation staining Treponema
Leptospirae
wall less Bacteria flourochrome Staining Mycoplasma
Rigid Thick walled Bacteria
Free Living (Extra cellular) Streptocooci
Gram positive Aerobic Bacillus Staphylococci

Cocci
Non aerobic Clostridium
Spore forming
Bacilli Filamentous Actinomyces
Non Spore forming Nocardia
Non filamentous Corynebacterium
Listeria
 Cocci Neissereia N.Meningitidis
N.Gonorrhea
Gram Negative
 Facultative Haemophillus influenza
Respiratory
Bordetella pertusis
Legionella pneumonia

Brucecella
Straight Zoonotic Francicella
Pasturella
yersenia

Enteric & related Esherrica Coli

Enterobacter
Gram Negative Bacilli Serratia
Klebsellia
Helicobacter Salmonella
Curved Campylobacter Shigella
Vibrio Cholera proteus

Aerobic Pseudomonas

Anaerobic Bacteroids
 Enteric & related

Lactose fermentaors Slow Lactose fermentaors Non Lactose fermentaors

Klebsiella Serratia Salmonella

E.Coli Vibrio Shigella
Enterobacter Proteus
pseudomonas

Gram Negative Bacilli

 Laboratory Diagnosis
• 1Sampling

• 2 Staining

Culture is Negative / can not be obtained

IgM/IgG
• 1. Detect antibody (Ab)in Pt’s specimen
HBsAg
• 2. Detect antigen (Ag) in Pt’s specimen
PCR
• 3. Detect Nucleic acids in Pt’s specimen
 Laboratory Diagnosis
• 1Sampling

• 2 Staining

• 3 Culture

• 4 Biochemical Reactions

• 5 Antimicrobial sensitivity
 Staining

Process of coloring the object / specimen( tissue or organisms)

Stains : these are salts used for staining.
1 Basic Stains
2.Acidic Stains
3.Neutral Stains
Human Cell
cytoplasm
Hematoxyline (Basic dye)

Nucleus Eosin (Acidic dye)

 Types of Stains
1 Basic Stains
Staining Component is Basic
Haematoxyline Basic fuchsin Crystal violte Methylene Blue
2.Acidic Stains
• Staining component is Acidic
Negrocin Indian ink Congo Red Eosin

3.Neutral Stains Giemsa Stain Leishman Stain

 Types of Stains
2 .on the Basis of Synthesis
• Synthetic Stains
• Most of stains are synthetic in nature
• Methylene Blue ,C.violet Basic Fuchsin ....
• Natural
• Hematoxyline , carmine
 Types of Staining

• 1. Negative Staining?? Background is stained Instead

of organism
• 2. Positive Staining organism is stained

• Simple staining(Direct) Only 1 stain is used

• Compound Staining(indirect) 2 or more stains are used

 Staining
Negative Staining Positive Staining
organism are stained
Background is stained
Instead of organism Simple Staining
(Direct Staining) Compound Staining
( Indirect Staining)
Only 1 stain is used used
> 1 stains are used applied
Crystal violet, Safranin etc
Regressive Staining
Progressive Staining
Decolourizer is not used Decolourizer is used

H&E Staining Gram’s Staining

Z N Staining
 Warthin -starry stain(Silver stain)

• Used to detect spirtochetes

• Bartonella henselae (cat scratch disease)
• H.pylori
• Legionella pneumoniae
Organisms appear dark brown & back ground appear as
Golden brown / golden yellow
 Negative staining
Stains the background instead of the organisms
as negative charge on cell wall of microorganisms
which repels the negatively charged stain
Dyes used in negative staining are acidic in nature
Negrosin
Indian ink
 Negative staining
used to view viruses,
bacteria
bacterial flagella
biological membrane
structures and proteins
protein aggregates
which all have a low electron-scattering power
Staining Process Appearance

Endospore Malachite green counterstained Endospores: Green Vegetative cells:

(Dornor's method) with Safranin Red

Capsule Copper Sulphate is applied A:Capsule: Light violet/ pale mauve

A: Hiss method (Positive color
technique) Bacteria: Purple
B: Manevals's technique
(Negative) B: Congo Red is applied B:capsule, bacterial cell, Stands out
against dark background

Cell wall Cell wall: Red

(Dyar's method) Cytoplasm: Blue
Flagella Flagella: Red
(Leifson's method) Vegetative cells: Blue
Corrynobacterium Metachromatic granules (Alberts's Granules: Bluish black, Cytoplasm:
Diptheria method) Green
 SIMPLE STAINING

• OBJECT: to study the morphological characteristics of

microorganisms
• Requirements:
• Distilled water
• staining tray
• Sample of microorganism

• Stain
• ( methylene blue ,safraninetc)
 BACTERIAL SMEAR PREPARATION
Smear
Thin layer of specimen spread over slide
Bacterial smear is prepared prior to conduct
simple stain, Grams stain or any other
• Requirements:
• Bacterial culture
• Distilled water
• Clean slide
• Permanent marker
• inoculating loop
• Staining tray
• Flame source slide warmer
 BACTERIAL SMEAR PREPARATION
Procedure
• STEPS
• 1 draw circle on slide & label it
• 2 place drop of water on the slide
• ( omit this step in case of liquid media)
• 3 Sterilize the wire loop till it becomes red hot
• Then cool it in air
• 4 Take sample by wire loop & spread it over slide
• starting from drop to periphery in circular way
• 5 air dry for 3 min
 Smear Fixation

• Process of fixing the Smear to slide

• Types
• Heat Fixation
• Alcohol fixation
• Purpose
• 1 To avoid floating of Bacteria & fixing with slide
• 2 killing the infectious organisms
• fix the slide by passing the slide over flame 3 times & then check it by
touching the back of slide to back of your hand to avoid over heating
 1 SIMPLE STAINING
• 1 prepare Bacterial smear of given specimen
• 2 put 2 to 3 drops of stain on bacterial smear and leave it
for 1 minute
• 3 wash it with distilled water then dry it
• 4Examine under microscope
• Observation
• Cocci appear Round
• Bacilli will appear Rod Shape
 2 GRAMS STAINING
was developed in 1884 by Danish physician christain Gram
It separates most bacteria in to 2 groups
Grams +ve which stain blue or purple
Grams –ve which stain red or pink
Grams stain is useful in 2 ways
1 Identification & Differentiating bacteria
2 influencing the choice of antibiotics because
Grams +ve bacteria are susceptible to penicillin
 GRAMS STAINING
Requirements:
1 Bacterial Sample
2 Glass Slide
3 Burner
4 Crystal violet dye (primary stain)
5 iodine solution (moderant)
6 Decolorizer acetone or Alcohol
7 Safranin (Counter Stain )
8 Cedar wood oil
9 Microscope
 Gram Staining procedure
•Make a smear & fix the smear to slide
•1 add crystal violet (primary stain ) over smear & leave it for 2-3 minutes to
retain the colour
•Rinse with D/W
•2 now add iodine on smear leave for a while
•3 rinse with with alcohol or acetone & than with water
•4 add Safranin ; leave for 2 minutes
•Observe under microscope
 GRAMS STAINING
PROCEDURE:
1 Prepare the Smear
2 Cover smear with few drops of crystal violet for 2-3 minutes
3 add Gram’s iodine wait for 2-3 min
4 wash the slide with alcohol at 45 degree then wash with water
5 counter stain by safranin leave for 30 sec
6 completely rinse the slide with water then dry it
7 Observe under microscope
 OBSERVATION/ Result
• Grams positive bacteriawill appear blue/purple because
they have thick mash like peptidoglycan 50 to 90% so
the crystal violet iodine complex will not leak out after
wash
• Grams negative bacteria will appear red/pink because
they are thin walled