Microscopic and macroscopic observation of microorganisms & Gram stain. Mgr. Tomáš Kastl

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1 Microscopic and macroscopic observation of microorganisms & Gram stain Mgr. Tomáš Kastl

2 MARKS TO NOTICE Morphology of colonies and cells - strructure - size - surface - shape - profile - special organels - edging AGE PLAYS THE MAIN ROLE!!! Color Growth in medium - native color of colonie - diffusal - cells after staining - sedimentary - flaky

3 Morphology of colony Staphylococcus aureus Bacillus subtilis Saccharomyces cerevisiea Sarratia maecenses

4 Morphology of cell Size Shape E. coli x S. cerevisiea

5 Color - mostly depends on the grownth medium - some microorganisms are named by their color: Staphylococcus aureus, Kucuria rosea...

6 Liquid medium Growth in medium Obligate aerobes Fermenting yeasts Moulds Aerobes Facultative anaerobes Aerobes Staphylococcus sp. some Bacillus sp. Facultative anaerobes Dead yeasts Lactobacillus sp.

7 TAXONOMY Bacterial species includes organisms, which possess similar genetic information - at least 70%. Recognition of genus and species 1 2 Enterococcus faecalis Domain Kingdom Phylum Class Order Family Genus Species Genus Species enterokokus x entheroccocuss

8 BACTERIAL CULTURE AND COLONY colony - population of one species, which overgrows maternal organism = clones of one cell culture - any microorganism cultivated in laboratory pure culture - one bacterial species mixed culture - few bacterial species

9 STAIN TYPES I. simple stain- visualisation of all cells II. negative stain - dyes only the background - observation of shape and size - indian ink III. vital stain - distinguish dead cells and living cells - only dead cells are dyed - tryphan blue negative stain vital stain

10 STAIN TYPES IV. differential stain - external stuctures (spores, membranes) - inner compounds (starch, glycogen) V. diagnostic stain - identification - Gram stain, Ziehl-Neelsen stain... Be sure your dye isn t toxic! differential stain

11 Diagnostic stain Gram stain - helps to deside, which treatment method to use G + some antibiotics affect sythesis and coupling of peptidoglycans disability create cell wall cell is exposed to osmotic lysis Hans Christian Gram

12 Diagnostic stain G - no peptidoglycans = no place where to act inhibitors of protein synthesis x inhibitors of cell wall s synthesis

13 Gram positive x gram negative

14 - similarity to gram positive bacteria - peptidoglycan layer is altered by layer of β-glucans

15 UNDYEABLE BACTERIA Borrelia burgdorferi Chlamydia pneumonie Mycobacterium tuberculosis Mycobacterium leprae Legionella dye crosses the cell wall in hot solution due their cell wall thikness and composition (mycolic acid, waxes...) are able retain zhe dye even after rinsing by acid ZIEHL-NEELSEN STAIN Mycobacterium tuberculosis

16 PROCEDURE Native preparate - rinse purefied slide glass in ethanol - dry it and pass throught the flame - in the middle of slide glass drop a drop of destil water - use anealed and recooled inoculation rod to transfer an appropriate amount of your culture to the drop - DO NOT SPREAD TI - just cover it with cover glass (be careful air bubbels are rather unwanted) - redundant water suck off with tissue - observe under the microskop cells are unaffected and alive it is possible to observe their natural movement

17 PROCEDURE Gram stain 1 - rinse purefied slide glass in ethanol - dry it and pass throught the flame - in the middle of slide glass drop a drop of destil water - carry out a steril transfer of your bacterial culture into the drop! be reasonable - STIR IT PROPERLY IN THE WATER - let it air - than pass the slide glass throught the un shinless patr of flame 3x for 4 second! WITH THE CELLS TURNING UPWARDS!

18 Gram stain 2 - bury this slide glass into the crystal violet for 30 secon - rinse it 2second in weak water carrent - than bury your simple into the Lugol's solution for 30 second - rinse it 2 second in weak water carrent again - apply ethanol but maximally 20 second - rinse it 2 second in weak water carrent again - dye cells with safranin for 1 minute - dry it and observe cells are dead and fixed to the slide glass

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