Lab 5 & 6 Staininig Tech & Unknowns Spring 2010-2
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Transcript of Lab 5 & 6 Staininig Tech & Unknowns Spring 2010-2
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Lab 6 & 7STAINING TECHNIQUES
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Different bacterial cells morphologies
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SPECIMEN PREPARATION FOR LIGHT MICROSCOPYPREPARATION OF A BACTERIAL SMEAR
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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
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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
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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
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Gram Stain ResultsGram negative rodsGram positive cocci
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DIFFERENTIAL STAINING TECHNIQUES Acid Fast stainMycobacteriumUnique waxy cell walls
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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
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SPECIAL STAINING TECHNIQUESNegative Staining for CapsulesCapsule Stain Results
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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)
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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.
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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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