Gram Staining & Endospore Staining PDF

Gram Staining & Endospore Staining  AIM OF THE EXPERIMENT  To learn the Gram Staining procedure and apply it correctly.  To be able to differentiate Gram (+) and Gram (-) bacteria.  To learn the mechanism of Gram Staining.  To learn about sporulation  To be able to stain endos...

Gram Staining & Endospore Staining  AIM OF THE EXPERIMENT  To learn the Gram Staining procedure and apply it correctly.  To be able to differentiate Gram (+) and Gram (-) bacteria.  To learn the mechanism of Gram Staining.  To learn about sporulation  To be able to stain endospores CELL WALL  DIFFERENCES GRAM (+) GRAM (-) Cell wall Thick Thin Peptidoglycan High amount Low amount Lipid Low amount High Amount Lipopolysaccharides No Yes Pores No Yes  STAINING PROCEDURE  Prepare the smear, (do not forget the fixation)  Cover the smear with Crystal violet (CV) for one minute  Rinse with water for 5 sec.  Cover the smear with iodine (I) for one minute  Rinse with water  Rinse with 95% ethanol solution dropwise  Cover the smear with safranin for 30 sec.  Rinse with water  Blot dry & put one droplet of oil on the smear and observe with microscope.  MECHANISM of STAINING  CV and iodine can pass through the bacterial cell wall.  CV-I complex ------ insoluble and big molecule  Alcohol break the lipids so CV-I complex can exit from the cell wall of Gram (-) bacteria  Alcohol cause dehydration of peptidoglycan layer so cell wall shrinks so CV-I complex cannot exit from the cell wall of Gram (+) bacteria  Safranin is used to colorized the Gram (-) bacteria  BACTERIA for today’s experiment Escherichia coli Bacillus subtilis Staphylococcus aureus Gram (-), rod-shaped Gram (+), rod-shaped Gram (+), grape-like clusters Why to Sporulate? Sporulation is the defense mechanism of some bacteria to survive in extreme environmental conditions and lack of nutrients. Spore formation enable bacteria to lie dormant for extended periods. Reversible: More favorable environmental conditions can cause germination. Properties of Endospores Endospores are thick walled, highly refractile and resistant form of the bacteria. Both G(+) and G(-) bacteria may form spore. There are many bacterial species forming spores (ex. Paenibacillus, Sporolactobacillus, Desulfotomaculum, Thermoanaerobacter, Sporomusa, Sporohalobacter, Anaerobacter, Alicyclobacillus, Amphibacillus, Heliobacterium, Heliophilum, Syntrophospora, Desulfitobacterium...) For food industry, our concern: Bacillus and Clostridium ssp. Endospore  Consists of: Resistant to:  DNA  Ribosome UV radiation  Dipicolinic acid (DPA) & Ca High temperature  Small acid-soluble proteins (SAS) Extreme freezing Chemical disinfectants Heat resistance of endospore is due to: Dehydration Ca-DPA complex ENDOSPORE vs VEGETATIVE CELL COMPARISON ENDOSPORE STAINING Culture: 48 h culture of bacteria Prepare heat-fixed smears Cover with malachite green & wait for 30 seconds Heat slides over the flame until steam is observed, add malachite green to prevent drying for 3 minutes Cool slides Rinse it with water Add safranin for 1 minute Rinse it Dry Oil immersion