Identification of bacteria using gram staining method

Staining

It is a process of observing, distinguishing, and identifying the bacteria with the help of a dye
or stain.

Stains and dyes are frequently used in biology and medicine to highlight structures in

biological tissues for viewing, often with the aid of different microscopes. Stains may be used
to define and examine bulk tissues (highlighting, for example, muscle fibers or connective
tissue), cell populations (classifying different blood cells, for instance), or organelles within
individual cells. In biochemistry it involves adding a class-specific ( DNA, proteins, lipids,
carbohydrates) dye to a substrate to qualify or quantify the presence of a specific compound.
Simple staining is staining with only one stain/dye.

Gram staining

Gram stain or Gram staining, also called Gram's method, is a method of staining used to

distinguish and classify bacterial species into two large groups (gram-positive and gram-
negative). The name comes from the Danish bacteriologist Hans Christian Gram, who
developed the technique.

This differential staining procedure separates most bacteria into two groups on the basis of
cell wall composition:

1. Gram positive bacteria (thick layer of peptidoglycan-

90% of cell wall)- stains purple

2. Gram negative bacteria (thin layer of peptidoglycan-

10% of cell wall and high lipid content) –stains red/pink
Some examples are,

Steps involve in gram staining:

1. Fixation of clinical materials to the surface of the microscope slide either by heating
or by using methanol. (# Methanol fixation preserves the morphology of host cells, as
well as bacteria, and is especially useful for
examining bloody specimen material).

2. Application of the primary stain (crystal

violet). Crystal violet stains all cells
blue/purple

3. Application of mordant: The iodine solution

(mordant) is added to form a crystal violet
iodine (CV-I) complex; all cells continue to
appear blue.

4. Decolorization step: The decolorization step

distinguishes gram-positive from gram-
negative cells. The organic solvent such as
acetone or ethanol, extracts the blue dye complex from the lipid-rich, thin walled gram-
negative bacteria to a greater degree than from the lipid poor, thick walled, gram-
positive bacteria. 

The gram-negative bacteria appear colorless and gram-positive bacteria remain blue.
 5. Application of counter stain (safranin): The red dye safranin stains the decolorized
gram-negative cells red/pink; the gram-positive bacteria remain blue.

Characteristics of gram positive and negative bacteria

Gram positive bacteria

Gram-positive bacteria are bacteria that give a positive result in the Gram stain test. Gram-

positive bacteria take up the crystal violet stain used in the test, and then appear to be purple-
colored when seen through a microscope. This is because the thick peptidoglycan layer in the
bacterial cell wall retains the stain after it is washed away from the rest of the sample, in the
decolorization stage of the test. Some other characteristics are following:

1. Cytoplasmic lipid membrane

2. Thick peptidoglycan layer

3. Teichoic acids and lipoids are present, forming lipoteichoic acids, which serve

as chelating agents, and also for certain types of adherence.
4. Peptidoglycan chains are cross-linked to form rigid cell walls by a bacterial enzyme DD-
transpeptidase.
5. A much smaller volume of periplasm than that in gram-negative bacteria.

Gram negative bacteria

Gram-negative bacteria are bacteria that do not retain the crystal violet stain used in the

gram-staining method of bacterial differentiation. They are characterized by their cell
envelopes, which are composed of a thin peptidoglycan cell wall sandwiched between an
inner cytoplasmic cell membrane and a bacterial outer membrane. Some other characteristics
are following:

1. An inner cell membrane is present (cytoplasmic)

2. A thin peptidoglycan layer is present (This is much thicker in gram-positive bacteria)

3. Has outer membrane containing lipopolysaccharides (LPS, which consists of lipid A, core

polysaccharide, and O antigen) in its outer leaflet and phospholipids in the inner leaflet
4. Porins exist in the outer membrane, which act like pores for particular molecules

5. Between the outer membrane and the cytoplasmic membrane there is a space filled with a
concentrated gel-like substance called periplasm
6. The S-layer is directly attached to the outer membrane rather than to the peptidoglycan

7. If present, flagella have four supporting rings instead of two

8. Teichoic acids or lipoteichoic acids are absent

Apparatus

For preparation of microbial colony

 Incubator
 Autoclave
 Petri-dish
 Aluminum foil
 Inoculation loop/needle
 Pipette
 Flask
 Measuring cylinder
 Agar
 Distilled water
 Micro-organisms from waste water

For Gram staining

 Glass slides
 Slide covers
 Waste water sample
 Microscope
 Inoculation loop
 Dropper

Chemicals are;

 Crystal violet
 Trapping agent (gram iodine)
 Decolorizer (Alcohol/ acetone)
 Counter strain (safranin)
Procedure

In this experiment, we have to make the microbial

colony on the agar first. Then we will do the gram
staining

Preparation of microbial colony

 Wash all the glass ware, petri dish, pipette,

cylinders, with distilled water.
 Wrap all the glass ware in aluminum foil to keep them away from sunlight.
 Put these wrapped glass ware in autoclave at 120°C for 15-20 minutes at 15 psi.
 Sterilize your hands or wear the surgical gloves.
 Weight 2gm of the agar. Dissolve it in 100ml of water in a flask and cover it urgently
with the cotton and aluminum foil.
 Now put it in autoclave at 120°C for 15-20 minutes at 15 psi.
 Now take 20ml of the sterilize agar media in a sterilize petri dish and make sure that
bottom is completely filled with the media.
 Now cover the petri dish to save it from contamination.
 Wait until the liquid media is cooled and converted into the jelly like substances.
 Inoculate the micro-organisms stains on the jelly of the media placed in the petri dish
with the sterilized inoculation needle.
 Incubate the prepare culture media with the micro-organism’s stains at 37°C
temperature for one day.

Gram staining procedure

First sterilize all the instruments in the autoclave at 115-120 degree Celsius at 15 pounds
pressure for 15-.20 minutes

 After growing the bacteria colony in the agar in the lab, pick one colony as a sample.
 Put the sample in water on the glass slide. Heat it slightly.
 Now stain the slide with crystal violet for 1 minute.
 Rinse the slide with distill water for 5 seconds.
Note: Rinsing should be done after every step of staining at the angle of 45 degrees.

 Now flood the slide with gram iodine for 1 minute.

 Rinse the slide with distill water for 5 seconds.
 Decolourize the slide by applying ethanol for 2-3 seconds.
 Rinse the slide with distill water for 5 seconds.
 Flood the slide with safranin(counter-stain) for 70-80 seconds
 Rinse the slide with distill water for 5 seconds.
 After it dry the slide, cover it with slide cover.
 Then observe the slide under the microscope and differentiate the bacteria that wether
it is gram
 negative or positive.

Observations

The bacterial colony that was observed under the microscope in the lab was gram-positive
bacteria.

Conclusions

From the experiment that we have done, finally, we can conclude that gram staining is
the method of distinguishing between gram-positive and gram-negative bacteria. In this
experiment, we were provided some material to help us for reaching the aim of this
experiment such as Crystal violet, Gram’s iodine, 95 % ethyl alcohol, safranin, and
microscope slide. However, before doing the experiment, we absolutely need to pay
attention on the precautions. There are several procedures that we have to do in order to
avoid the error in this experiment, such as prepare smear from cultures of microorganism,
heat fix the smears, place the slides on a staining rack, and so on.