Environmental Engineering Department
Environmental Microbiology Lab
By Dr. Nayab Zahra
University of Engineering and Technology, Taxila
“Identification of Bacteria by Gram
Staining Process.”
Introduction
Microbial Staining – Giving color to microbes.
Because microbes are colorless and highly transparent
structures.
Staining – Process in which microbes are stained.
Introduction
Stains/dyes - Organic compounds which carries either
positive charges or negative charges or both.
Based on the charges:
❑ Basic stain/dyes – stain with +ve charge.
❑ Acidic stain/dyes – stain with –ve charge.
❑ Neutral stain/dyes – stain with both charges.
Introduction
Based on function of stain:
Simple staining – Only one dye is used- differentiation
among bacteria is impossible- Eg. Simple Staining.
Differential staining - More than one dye is used-
Differentiation among bacteria is possible- Eg. Gram’s
staining, Acid-fast staining.
Special staining – More than one dye used -Special
structures are seen. Eg. Capsule staining, Spore staining.
GRAM STAINING
DANISH BACTERIOLOGIST HANS CHRISTIAN
GRAM (1884).
Based on this reaction, bacteria classified into
Gram positive and Gram negative bacteria.
The cell wall composition differences makes
difference.
Gram +ve and Gram -ve Bacteria
Gram positive bacteria (thick layer of
peptidoglycan-90% of cell wall)- stains Purple.
Gram negative bacteria (thin layer of
peptidoglycan-10% of cell wall and high lipid
content) –stains Red/Pink.
• Inner most plasma membrane • Inner most plasma membrane
• Thin peptidoglycan cell wall • Thick peptidoglycan cell wall
• Another plasma membrane • Outer capsule
• Outer capsule • More easily treatable with
• Harder to treat with antibiotics
antibiotics. • Stain purple after Gram stain
• Stain pink after Gram stain
Environmental
Significance
Environmental Significance
Water quality scientists may use the Gram stain as a
confirmatory tool for the detection of fecal bacteria in water
samples.
For clinical microbiology applications, the Gram stain is used to
confirm the identity of bacteriological disease agents along
with traditional diagnostic methods.
Environmental Significance
For environmental microbiologists, Gram stain helps in the
categorization of bacterial populations according to cell wall
structure.
Fast, inexpensive and cheap way to define type of cell wall.
Apparatus Apparatus
Microscope Glasswares Slides Tweezers
Cover slips Tooth picks Dropper Inoculation loop
Chemicals
Crystal Violet - Primary stain
Gram’s iodine
Alcohol / Acetone (95%)- decoloriser
Safranin
PRINCIPLE
Crystal violet - All bacteria take crystal violet- so all appears
violet.
Iodine – Crystal Violet-iodine(CV-I) complex is formed.
Acetone - Bacteria with high lipid content loose CV-I
complex (appear colorless) but bacteria with less lipid
content retains CV-I complex ( appear violet).
Safranine/ dilute carbol fuchsin – Only colorless bacteria
takes – appear pink.
PRINCIPLE
When stained with a primary stain some bacteria are able to
retain the primary stain by resisting declorization while
others get decolorized by a decolorizer. Those bacteria
which retain the primary stain are called Gram +ve and
those bacteria which gets decolorized and then get
counterstained are called Gram -ve.
Procedure
Smear preparation :
Smear - is a distribution of bacterial cells on a slide for the
purpose of viewing them under the microscope
Method:
❖ Take a grease free dry slide.
❖ Sterilize the inoculating loop on a flame of a Bunsen burner.
Procedure
❖ Transfer a loop full of culture (or the specimen) by sterile loop
and make a smear at the center. Smear should not be very
thin or very thick.
❖ Allow the smear to dry in the air.
❖ Fix the dry smear by passing the slide 3-4 times through the
flame quickly with the smear side facing up.
❖ The smear is now ready for staining.
Procedure
Place the slides on the staining rods.
Cover the smear with crystal violet stain and leave for 1
minute.
Wash carefully under running tap water.
Flood the smear with Gram’s iodine solution and leave for 1
minute.
Drain off the iodine. Wash the slide for the again in a gentle
stream of tap water.
Procedure
Flood the slide with the decolorizing agent then wait for 20-30
seconds. This can also be done by adding a drop by drop to
the slide until the decolorizing agent running from the slides
runs clear.
Gently wash the slide under running tap water and drain
completely.
Counterstain with safranin and wait for about 30 seconds to 1
minute.
Procedure
Wash slide in a gentile and indirect stream of tap water until
no color appears in the effluent and then blot dry with
absorbent paper.
Observe under microscope.
Procedure
Procedure
Common Error causes:
❑ Excessive heating
❑ Less concentration of crystal violet
❑ Excessive washing
❑ Insufficient exposure to iodine
Observation