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