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Lab 6 & 7

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 SIMPLE STAINING TECHNIQUES

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Lab 6 & 7

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  1. Lab 6 & 7 STAINING TECHNIQUES

  2. Different bacterial cells’ morphologies

  3. SPECIMEN PREPARATION FOR LIGHT MICROSCOPY • PREPARATION OF A BACTERIAL SMEAR

  4. SPECIMEN PREPARATION FOR LIGHT MICROSCOPY • PREPARATION OF A BACTERIAL SMEAR • FIXING • STAINING • SIMPLE STAINING TECHNIQUES • DIFFERENTIAL STAINING TECHNIQUES • Gram Stain (1884-Hans Gram) • Capsule Staining • Endospore Stain • Acid fast staining

  5. DIFFERENTIAL STAINING TECHNIQUESGram Stain Groups the bacteria into: Gram Positives: Resistant to decolorization, retain primary satin Gram Negatives: Cells decolorize, so take the counterstain Primary stain Mordant Acetone-Alcohol Counterstain

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

  7. Gram Stain Results Gram negative rods Gram positive cocci

  8. DIFFERENTIAL STAINING TECHNIQUES • Acid Fast stain • Mycobacterium • Unique waxy cell walls

  9. 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

  10. SPECIAL STAINING TECHNIQUES • Negative Staining for Capsules • Capsule Stain Results

  11. 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)

  12. 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.

  13. 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

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