Lab 6 & 7STAINING TECHNIQUES

Different bacterial cells morphologies

SPECIMEN PREPARATION FOR LIGHT MICROSCOPYPREPARATION OF A BACTERIAL SMEAR

SPECIMEN PREPARATION FOR LIGHT MICROSCOPYPREPARATION OF A BACTERIAL SMEARFIXINGSTAINING

SIMPLE STAINING TECHNIQUES

DIFFERENTIAL STAINING TECHNIQUES Gram Stain (1884-Hans Gram) Capsule Staining Endospore Stain Acid fast staining

DIFFERENTIAL STAINING TECHNIQUES Gram StainGroups the bacteria into: Gram Positives: Resistant to decolorization, retain primary satin Gram Negatives: Cells decolorize, so take the counterstain

Primary stainMordantAcetone-AlcoholCounterstain

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 ResultsGram negative rodsGram positive cocci

DIFFERENTIAL STAINING TECHNIQUES Acid Fast stainMycobacteriumUnique waxy cell walls

SPECIAL STAINING TECHNIQUESNegative Staining for CapsulesCapsulesDeterminants for virulence in some genera of bacteriaCapsule StainingPreparation of a bacterial smear along with negative staining w/ Nigrosin or India InkCounterstain w/ Crystal Violet

SPECIAL STAINING TECHNIQUESNegative Staining for CapsulesCapsule Stain Results

STAININGSPECIAL STAINING TECHNIQUESEndospore StainGenera of bacteria that produce endosporesBacillus & ClostridiumOther genera: Desulfotomaculum, Sporosarcina, Sporolactobacillus, Oscillospira, ThermoactinomycesSchaeffer-Fulton Method for Endospore StainingPreparation of a Bacterial Smear-Air dryApplication of Primary Dye (Malachite Green) w/ concurrent application of heatAllow slide to coolRinse w/ waterApplication 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 7Ex. 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. 543Ex. 3-10 Endospore satin. Organism Bacillus cereus. Procedure in pg. Report in pg. 545. Q. 1

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