Pharmaceutical Microbiology I

Preparation of Smears, simple staining

and Gram staining
Learning Outcomes

1. Prepare and fix a smear.

2. List the advantages of staining microorganisms.

3. List the different bacterial staining methods.

4. Explain the basic mechanism of staining.

5. Perform a simple direct stain.

6. Explain the rational and procedure for the gram stain.

7. Perform and interpret Gram stains.

Background
 Simple stain: only one reagent is used and all bacteria are usually stained similarly.

basic stain (positive ion) and acidic stain (negative ion)

 Differential stain: Multiple reagents are used and bacteria react to reagent differently.
Ex: Gram stain

 Structural stain: used to identify specific pats of microorganisms

 To stain bacteria, a thin film of bacterial cells, called a smear, must be placed on a slide.

First steps in identifying Microorganisms ???

 Simple Stain
(Methylene blue)

Differential stain
(Gram Stain)
Bacterial stains
Basic staining
Methylene blue, safranin,
Simple Staining carbolfuchsin, crystal violet
To observe shape and arrangement
of bacterial cells
Acidic staining
(Eosin, nigrosin)

Staining
Gram staining
Separation into groups
Acid-Fast staining
(Ziehl-Neelsen)

Differential Staining
Spore staining

Visualization of specific structures Capsule staining

of bacteria
Flagella staining

Inclusion staining
Watch the video
Gram Staining
 Primary stain (Crystal violet)
 Mordant (Gram`s Iodine)
 Decolorizer agent (ethanol)
 Secondary stain / counterstain (safranin)
 Gram Staining Procedure
KEY

Crystal violet
Iodine
Alcohol
Safranin

Gram-positive
Gram-negative

1 Application of 2 Application of 3 Alcohol wash 4 Application of

crystal violet Iodine (mordant) (decolorization) safranin (counterstain)
(purple dye)

Iodine reacts with Alcohol dehydrate the Safranin stain only

the CV dye in the GP cell wall and CV-I decolorized cell wall
cytoplasm and form crystal remain inside. bacteria into red.
a CV-I crystal that However, in GN cell
remain inside the wall, alcohol dissolve
bacteria. the LPS layer and wash
out the CV-I crystal.
Gram Staining
Gram staining : Introduction
 Gram’s procedure divides bacterial organisms into two broad groups:

 Gram-positive : bacteria appear purple after a Gram staining procedure.

 Gram-negative : organisms appear pink after a Gram staining procedure.

 Gram staining is the first step in identifying bacteria.

 Bacteria that resist decolorization by 95% ethanol are arbitrarily termed Gram positive and
those that do not are Gram negative

 The characteristic compound found in all true bacterial cell walls is peptidoglycan. The
amount of PPG is among one of the differences between the GP and GN cell walls.
Bacterial Cell Wall: Gram + versus Gram -
Helpful Tips for Gram staining
Several conditions should be followed for a valid Gram staining procedure: 

1. Young cultures (less than 24 h)

- Because when bacteria die, their cell wall degrade and may retain the primary stain giving inaccurate
results.

2. Thin smear
-Thicker or uneven smears will result in uneven staining and decolorization.

3. Fresh reagents
- Avoid using old or expired reagents. Reagents should still have their proper strength without any
crystal intrusions.

4. Overheating :
- Avoid the overheating during the cells fixation ( during the smear preparation)

5. Respect the timing of each step

- Allowing enough time for each stain to sit or not allowing too much time for decolorizer and/or water to
sit on slide.

6. Control cultures
- known GP bacterium and GN culture.
To the practical work
Procedure 1: Preparation of Bacterial Smear

Procedure 2 : Smear staining

Procedure 3 : Gram staining

Needed material
 S. aureus agar fresh culture
 E. coli agar fresh culture
Nutrient
 L. gasseri agar fresh culture Agar Water bottle Bunsen burner

 Stain rack
 Bibulous paper
 wash bottle [marked H2O]
 Lens paper Microscope slide Lens paper Inoculating loop
 Microscope slides
 Inoculating loop
 Bunsen burner
 Striker (to light Bunsen burner)
Clothes pin Grease pencil
 Grease pencil Methylene blue stain

 Methylene blue stain

 Gram stain kit
 Clothes pin
 Immersion oil

Gram stain Immersion oil Bibulous paper

Preparation of Bacterial Smear (1)
Preparation of Bacterial Smear (2)
Procedure 1 : Smear preparation
 Helpful Tips for Smear
preparation
1. A thin film of bacteria should be spread upon the slide. (If the smear is too thick, it is difficult to see
anything because there will be little light passing through).

2. The smear should be allowed to dry.

3. Once the smear has dried, the slide should be passed over a lit Bunsen burner several times to affix
the organisms. (This procedure is known as heat fixing.)

4. There is a slight shrinkage of cells during this process which is normal, but it helps the bacterial cells
to adhere to the slide through several rinses.

5. If the slide is overheated, the cells will warp and structure will be indistinguishable. (If heat is
applied to the cell before the smear is dry, there will be distortion.)

6. A properly stained bacterial smear should be slightly difficult to see to the naked eye. If there are
dark splotches of color, the bacteria are piled on top of each other.
Procedure 2 : Smear staining
Procedure 3 : Gram staining
E. coli _Nutrient Agar S. aureus _MSA Agar

Procedure 1:
Smear preparations

Slide 1 Slide 2

Procedure 2: Simple staining

(Methylene blue)

Do not forget
Microscope observation to label the
slide

Results Interpretation : Shape ? Arrangement? Size ??

S. aureus E. coli
E. coli _NA
Lactobacillus gasseri _MRS S. aureus _MSA

Procedure 1: Smear preparations

Slide 3 Slide 4 Slide 5

Procedure 3: Gram staining

Do not forget
Microscope observation to label the
slide

Results Interpretation : Shape ? Arrangement? Size ?? And gram + or - ?

S. aureus L. gasseri
Correction of the lab report
(Exercise 3)