This document provides an overview of procedures for examining stained microorganisms, including smear preparation and simple and Gram staining. It discusses the steps of inoculation, isolation, incubation, inspection, and identification of microbes in a laboratory setting. Specifically, it outlines the Gram stain procedure, which uses two dyes and a decolorization step to differentiate bacteria based on their cell wall composition into Gram-positive or Gram-negative categories. The Gram stain is a commonly used differential staining technique and first test in bacterial identification.

1. Medical microbiology
Lab 2: Examination of stained
microorganisms: smear preparation and
simple staining, Gram staining
Asst. Lec. Mariam A. Ali
Asst. Lec. Iman. k. kadhim
Al-Mustaqbal University college 2 st Class, 1 st Semester
Department of pharmacy

2. 1-Inoculation: Producing a pure culture
2-Isolation: Colony on media, one kind of microbe,
pure culture
3-Incubation: growing microbes under proper
conditions
4-Inspection: Observation of characteristics (data)
5-Identification: use of data, correlation, to identify
organism to exact species

3. 1-Inoculation: Producing a pure culture
Introduce bacteria into a growth medium using “aseptic” technique to
prevent contamination. Tools: Bunsen burner, loop. Needle, etc.
In working with microorganisms, we must have a method of
transferring growing organisms from a pure culture to a sterile
medium without introducing any unwanted outside contaminants.
This method of preventing unwanted microorganisms from
gaining access is termed aseptic technique.

4. 2-Isolation: Colony on media, one kind of
microbe, pure culture: isolation on general and
special “differential media”
General growth media: NA, TSA
Differential: Mac, EMB, SS , These have
dyes, salts, inhibiting agents.
Gram – bacilli, green
sheen on EMB: E.coli

5. 1
3
1
2
4

6. • 3- Incubation: Allow organisms to grow under the
optimal conditions
• Temperature, with or without oxygen etc.

7. • 4- Inspection: Observation, description
• Colony Morphology, Microscopic examination (grams stain)
• Systematic recording of “DATA”

8. • 5-Identification: Correlating data
from all observations to identify
organism to species
• Ex., green sheen on EMB,
• ferments lactose, Gram – bacilli,
• :E.coli.
Gram + bacilli
Gram - bacilli

9. Staining bacteria cells
Staining bacteria cells for microscopic examination makes it
possible
• to define their cell size, shape, arrangement;
• to study their chemical properties,and structures.
These characteristics can be use for
bacterial identification

10. Overview of a bacterial
staining procedure

11. Simple stains use a single basic dye (e.g. crystal
violet, methylene blue, safranin) to color bacterial cells
so that their size, shape and arrangement can be
observed
Staining bacteria cells:simple staining
one dye

12. •Differential stains, such as the Gram stain and the acid-fast
stain, differentiate bacteria based on the chemical
composition of their cell wall.
•Differential stain use two dyes instead of one: the first stain
is the primary stain, the second is the counterstain.
•A decolorization step occurs between the application of the
primary stain and counterstain.
•Depending on the composition of the cell wall, bacteria will
either retain the primary stain during decolorization or lose
the primary stain and take up the counterstain.
Staining bacteria cells:
differential stain
two dyes
complex procedure, see difference between cells

13. GRAM STAIN PROCEDURE
1. Stain with crystal violet 2%…… ……….….1 min.
2. Gram’s iodine (Lugol)………………………1 min.
3. Wash off with tap wate.
4. Decolorizer (Alcohol 50%-Acetone 50%)…20 sec.
5. Wash off with tap water
6. Safranin 0,25%………………………………1 min.
7. Wash off with tap water
8. Blot dry with bibulous paper
•Hans Christian Gram was a Danish
bacteriologist. He developed the Gram stain
as a means to differentiate pneumococci
from Klebsiella pneumonia in 1884.
•The Gram stain is often the first test
performed in the identification of bacteria.

14. 1

16. Overview of the Gram stain
Gram + Gram -
Staphylococcus aureus