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Microscopic and macroscopic observation of
microorganisms
&
Gram stain
Mgr. Tomáš Kastl
MARKS TO NOTICE Morphology of colonies and cells
- strructure - size
- surface - shape
- profile - special organels
- edging
Color Growth in medium
- native color of colonie - diffusal
- cells after staining - sedimentary
- flaky
AGE PLAYS THE MAIN ROLE !!!
Morphology of colony
Staphylococcus aureus
Saccharomyces cerevisiea
Bacillus subtilis
Sarratia maecenses
Morphology of cell
Size Shape
E. coli x S. cerevisiea
Color
- mostly depends on the grownth medium
- some microorganisms are named by their color: Staphylococcus aureus, Kucuria rosea...
Growth in medium
Obligate aerobes
Fermenting yeasts
Moulds
Aerobes
Facultative anaerobes
Aerobes
Staphylococcus sp.
some Bacillus sp.
Facultative anaerobes
Dead yeasts
Lactobacillus sp.
• Liquid medium
TAXONOMY Domain
Kingdom
Phylum
Class
Order
Family
Genus
Species
Bacterial species includes organisms, which
possess similar genetic information - at least
70%.
Recognition of genus and species
enterokokus x entheroccocuss
Enterococcus faecalis
Genus Species
1 2
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
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 vital stain
negative stain
IV. differential stain
- external stuctures (spores, membranes)
- inner compounds (starch, glycogen)
V. diagnostic stain
- identification
- Gram stain, Ziehl-Neelsen stain...
STAIN TYPES
differential stain
Be sure your dye isn´t toxic !
Diagnostic stain
Gram stain - helps to deside, which treatment method to use
Hans Christian Gram
some antibiotics affect sythesis and
coupling of peptidoglycans
→ disability create cell wall
→ cell is exposed to osmotic lysis
G+
Diagnostic stain
G-
inhibitors of protein synthesis x inhibitors of cell wall´s synthesis
no peptidoglycans =
no place where to act
Gram positive x gram negative
- similarity to gram positive bacteria
- peptidoglycan layer is altered by layer of β-glucans
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
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
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 !
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