Introduction to Lab Ex 7. Differential Stains Gram Staining, Endospore Staining,
Introduction to Lab Ex. Differential Stains – Gram Staining
Basic classification of bacteria is based on the cell wall structure.
There are 2 main groups: Gram positive and Gram negative.
Gram staining is a differential staining technique that provides an easy differentiation of bacteria into one of two groups.
The staining technique, developed in the late 1700’s by
Christian Gram classifies the rigid cell walled bacteria into one of
two groups based on whether they are able to resist the decolorizing
action of an alcoholic solution.
Those that resist decolorization by 95% ethanol are arbitrarily termed Gram positive and those that do not are Gram negative
• Thick peptidoglycan • 90% peptidoglycan • Teichoic acids
• 1 layer
• Not many
polysaccharides • In acid-fast cells,
contains mycolic acid
Gram-positive cell walls Gram-negative cell
walls
• Thin peptidoglycan • 5-10% peptidoglycan • No teichoic acids
• 3 layers
• Outer membrane has lipids, polysaccharides • No acid- fast cells
(mycolic acid)
The characteristic compound found in all true bacterial cell walls is peptidoglycan. The amount of PPG is among one of the
The process includes the use of:
a primary stain (crystal violet)
a mordant (helper) iodine solution, a decolorizer (95% ethanol),
The Gram stain
The Gram stain
Thin smear/heat fix
Gram stain:
a. Flood slide with crystal violet and let stain for 1 minute.
b. Drain off crystal violet and rinse off with distilled water; flood slide with Gram's iodine for 1 minute.
c. Rinse off Gram's iodine with distilled water.
d. Hold the slide on an angle (preferably with a clothes pin) and drop 95% ethyl alcohol onto it until the alcohol leaving the slide no longer has a purple tint; be sure to drop the alcohol onto the upper portion of the slide so that the smears are subjected to uniform decolorization. Be careful not to "decolorize" dye from the clothes pin!!
e. Rinse with distilled water and flood the slide with safranin and let stain for 2-3 minutes.
The crucial step in the staining process is the decolorizing step.
The most accepted theory about the rationale for the Gram staining
process is the one proposed by Salton.
This theory relies on the fact that the PPG is found in layers and the stain molecules are trapped within the many layers of the GP CW
when they form the complex with the mordant Iodine molecules.
Since the GN CWs lack much PPG the amount of stain captured in those CWs is much lesser.
When the cells are treated with the decolorizer – the ethanol – this
causes denaturation of the proteins in the outer membrane of the
GN CWs resulting in gaping holes in these CWs that lead to the
removal of the crystal violet-iodine complexes easily, leaving these cells unstained.
There are 4 conditions to be followed for a valid Gram staining procedure:
Young cultures - must be young within 18-24hrs old
(older cultures lose their Gram staining properties due to changes in the CWs as the cells get older)
Thin smear
thicker or uneven smears will result in uneven staining and decolorization
Fresh reagents - of proper strength
Demos: Gram stained slides of
Neisseria, Streptococcus, Pseudomonas, Actinomyces species.
Neisseria
Pseudomonas
Some bacterial strains can generate endospores when starved for nutrients as a mechanism of survival.
An endospore is a dormant and highly resistant cell which
contains a copy of the cellular DNA. Endospores germinate into vegetative cells when nutrients are around.
Endospores are often resistant to: -drying/desiccation
-high temperature -radiation
-chemical damage
Revival of endospores millions of years old has been claimed
(bees in amber, tombs of Egyptian Pharaohs)
http://micro.cornell.edu/cals/micro/research/labs/angert-lab/bacterialendo.cfm
Spore Stain Steps
Spore
Spore
producer
producer Spore non-Spore non-producerproducer before before staining staining stain with stain with Malachite Malachite green green decolorize decolorize (water) (water) counterstain counterstain (safranin) (safranin)
adapted from Leboffe & Pierce (2002) Fig. 3-50
adapted from Leboffe & Pierce (2002) Fig. 3-50 http
III.
Acid-fast bacterial cell
wall
http://www.chemsoc.org/chembytes/ezine/images/ 1998/evans_fig2.jpg
III. Acid-fast Stain
medlib.med.utah.edu/WebPath/ TUTORIAL/MTB/MTB013.html
Mycobacteria colonies
Mycobacterium tuberculosis
Mycobacterium tuberculosis
III. C. Acid-fast technique
acid-fast
species
non acid-fast
species
1. carbolfuschin
2. acid-alcohol
Todays Lab
Differential staining
Grams stainPositive control : Staphylococcus aureus
Negative control : Escherichia coli
Unknowns : Bacillus subtilis (1)and Gum smear (2)
Mix slide (3) E. coli and S. aureus (one loop-full each) Do a separate slide for each unknown (three of them), each slide needs BOTH positive and negative controls along with the unknown
Endospore Stain: Bacillus subtilis (1 Slide per student) (Follow protocol in Lab Manual)
Acid Fast Stain: Mycobacterium smegmatis (one slide per student) (Follow Protocol in Lab Manual)
