Lab 6 & 7

STAINING TECHNIQUES

Different bacterial cells morphologies

SPECIMEN PREPARATION
FOR LIGHT MICROSCOPY
PREPARATION OF A BACTERIAL
SMEAR

SPECIMEN PREPARATION FOR LIGHT

MICROSCOPY
PREPARATION OF A BACTERIAL SMEAR
FIXING
STAINING
1. SIMPLE STAINING TECHNIQUES
2. DIFFERENTIAL STAINING TECHNIQUES

Gram Stain (1884-Hans Gram)

Capsule Staining

Endospore Stain

Acid fast staining

DIFFERENTIAL STAINING TECHNIQUES

Gram Stain
Groups the bacteria into:
Gram Positives: Resistant to decolorization, retain primary satin
Gram Negatives: Cells decolorize, so take the counterstain

Primarystain
Mordant
AcetoneAlcohol
Counterstain

That behavior is due to differences in the composition of bacterial

cell wall, establishing 2 major groups of bacteria according to
their cell wall structure (Gram + and Gram - bacteria)

Gram-positive bacteria are encased in a plasma

membrane covered with a thick wall of peptidoglycan.
Gram-negative bacteria are encased in a triple-layer.
The outermost layer contains lipopolysaccharide.
Pepticoglycan is much thinner

 Gram Stain Results

Gramnegativerods

Grampositivecocci

DIFFERENTIAL STAINING TECHNIQUES

Acid Fast stain
Mycobacterium
Unique waxy cell walls

 SPECIAL STAINING TECHNIQUES

Negative Staining for Capsules
Capsules
Determinants for virulence in some genera of
bacteria
Capsule Staining
Preparation of a bacterial smear along with
negative staining w/ Nigrosin or India Ink
Counterstain w/ Crystal Violet

 SPECIAL STAINING TECHNIQUES

Negative Staining for Capsules
Capsule Stain Results

STAINING
SPECIAL STAINING TECHNIQUES
Endospore Stain
Genera of bacteria that produce endospores
Bacillus & Clostridium
Other genera: Desulfotomaculum, Sporosarcina,
Sporolactobacillus, Oscillospira,
Thermoactinomyces
Schaeffer-Fulton Method for Endospore Staining
Preparation of a Bacterial Smear-Air dry
Application of Primary Dye (Malachite Green) w/
concurrent application of heat
Allow slide to cool
Rinse w/ water
Application of Counterstain (Safranin)

PROCEDURES LAB 6
1) GRAM STAIN: EX. 3-7. Procedure in pg. 108-109,
Organisms: Staphylococcus aureus (Sa); Pseudomonas
aeruginosa (Pa) .
(Both in one slide)
REPORT IN PG. 539-540 (SKIP QUESTION 3 & dimensions)
2) Acid fast stain: Ex. 3-8. cultures: Mycobacterium
smegmatis (Ms) & Staphylococcus aureus (Sa) USE
PLATES . Procedure in Pg. 111-112). REPORT IN PG. 541
3) Start working with your Unknown:
Streak your unknown culture on 2 NA plates, using 4 phase streak
plate technique. Incubate 1 NA plate at 37 C and the other one at 30 C.
Read Ex.4-5, 4-6, 4-7 for theory on these media. AND EX. 5-31 FOR
INFORMATION on unknowns identification. You may also read
Chapters 3-14, 7-7, 7-8 & 7-9 as a background for this assignment.

Procedures Lab 7
Ex. 3-9: Capsule stain. Organism: Klebsiella
pneumonia. Procedure in Pg. 115-116.
Step 1: Instead of serum and Congo Red, we
use India Ink : Mix 1 drop of saline with a loopful
of the organism, and add one drop of India Ink
Step 7: Instead of Maneval, we use Cristal Violet
Report on Pg. 543
Ex. 3-10 Endospore satin. Organism Bacillus
cereus. Procedure in pg. Report in pg. 545.
Q. 1