lab manual 14-18

what are bacteria stained before viewing them under a microscope

to increase contrast and make them easier to see

what is a simple stain

a staining technique that uses one stain

what are the two types of simple stains

direct and indirect

what happens during direct simple staining

the stain colors the cells

what happens during indirect simple staining

the stain colors the background, not the cells

what is the stain used in the simple stain procedure in lab 14

methylene blue

what is the purpose of heat fixing a smear

to inactivate cellular enzymes and help cells adhere to the slide

when should a smear be heat fixed?

after it has completely dried.

how should a smear from solid media be prepared

mix a small amount of bacterial growth with a drop of water

how should a smear from broth culture be prepared?

agitate the broth, then transfer 1-2 drops to the slide aseptically

why should the broth be agitated before making a smear

to suspend the bacterial cells evenly

under what magnification are simple stained bacteria observed in this lab?

1000x oil

what is the purpose of lab 14: simple stains

the purpose to observe cells that are usually transparent by staining them and creating contrast.

what is a negative stain

a stain that colors the background rather than the cells.

what substance is used to create the dark background in a capsule stain?

india ink

why is a negative stain smear not heat fixed

to preserve the cells’ natural morphology and size

• only air dry WITH the stain

which stain is added after india ink in lab 15

crystal violet or safranin

what is the purpose of the counterstain in lab 15 negative staining?

to add color to the bacterial cells for better contrast.

glycocalyx

a gelatinous polymer made up of polysaccharide, polypeptides, or both that lies externally to the cell wall and can form a capsule or a slime layer.

• protects the cell and helps it attach to surfaces.

capsule

a type of glycocalyx that is firmly attached to the cell wall, protecting bacteria from phagocytosis, prevents dessication, and adhering to surfaces.

• it is associated with pathogenic strains of bacteria.

endotoxin

a toxic lipid components of the lipopolysaccharide of gram negative bacteria that is released when bacteria multiply or undergo lysis.

why are capsules often associated with pathogenic bacteria

it helps bacteria evade host defenses and establish infections

why might a bacterial culture lose its ability to produce capsules?

capsule production may be lost after several generations in lab culture

• this is why it shouldn’t be used for making major decisions concerning classification of bacteria.

• biochemical testing is more reliable.

who developed the gram stain and when

hans christian gram in 1884

what type of staining technique is gram staining

differential staining

what is the purpose of gram staining

to differentiate bacteria based on differences in their cell walls

why is gram staining useful when identifying an unknown bacterium

it is usually the first step of bacterial identification

why is gram staining useful to physicians

it helps guide antibiotic selection because antibiotic activity varies with cell wall type.

what color are gram positive bacteria

purple or dark blue

what color are gram negative bacteria

pink or red

why do gram + cells remain purple after decolorization

they retain the crystal violet primary stain

why do gram - cells become pink or red

they lose crystal violet during decolorization and absorb safranin

what is the primary stain in gram staining

Gram’s crystal violet

what is the mordant in gram staining?

gram’s iodine

what is the decolorizer in gram staining?

95% alcohol

what is the counterstain in Gram’s staining

gram’s safranin

why must decolorization be performed carefully

over or under decolorization can cause incorrect gram stain results

why should a young bacterial culture be used for gram staining?

older gram positive cells may lose their ability to retain crystal violet

how old should a culture be for gram staining?

24 hours old or younger

what does gram variable mean

both gram positive and gram negative staining reactions appear in the same field

Which genus may appear Gram-variable because of its waxy cell walls?

Mycobacterium.

what unusual gram staining pattern can occur in bacillus megaterium

Both Gram-positive and Gram-negative areas can appear within one cell.

how should the smear be prepared before gram staining?

air dry completely, then heat fix

what is an endospore

a dormant, thick walled, highly resistant structure produced by certain bacteria

what is the main function of an endospore

to help bacterium survive unfavorable conditions

which bacterial phylum contains many endospore producing bacteria

firmicutes

what conditions can endospores survive?

heat, UV radiation, toxic substances, and lack of water or oxygen.

how long can endospores remain viable?

years, centuries, or longer

why are endospores difficult to stain

their resistant protective endospore coat prevents ordinary stains from entering easily.

what is the hot malachite green method also called

schaeffer-fulton method

what is the primary stain in the hot malachite green method

malachite green

what is the counterstain in the endospore stain

safranin

why is the slide steamed during endospore staining

heat helps drive malachite green into the resistant endospore coat

how long do you steam slides for?

10 min/

what color are vegetative cells after endospore staining

red or pink

what color are endospores after staining

green

what are vegetative cells

the active, growing cells of bacteria

Why is safranin added after malachite green?

To stain vegetative cells and provide contrast with green endospores.

What do endospores look like under the microscope?

Highly refractive, regularly sized oval structures.

what is the alternative simple stain mentioned for visualizing endospores

methylene blue

How are endospore-stained slides prepared before steaming?

Prepare a smear, air-dry it, and heat-fix it.

what type of staining technique is acid fast staining

differential staining

what is the purpose of acid fast staining?

to identify bacteria with mycolic acids in their cell walls.

what are mycolic acids

waxy lipids found in certain bacterial cell walls.

which important bacterial groups are associated with mycolic acids

mycobacterium and nocardia

why are these bacteria called acid fast staining?

they retain the primary stain after acid alcohol decolorization

what is the primary stain in acid fast staining?

carbolfuchsin

what is the decolorizer in acid fast staining

acid alcohol solution

what is the counterstain in acid fast staining

methylene blue

what color are acid fast bacteria

red

what color are non acid fast bacteria

blue

why do acid fast bacteria retain carbolfuchsin

their waxy, mycolic acid containing cell walls retain the primary stain because carbolfuchsin is lipid soluble.

which diseases can be caused by acid fast organisms

leprosy, nocardiosis, tuberculosis

what two organisms are used in lab 15

mycobacterium smegmatis

alcaligenes faecalis

why must mycobacterium be spread carefully on the slide for acid fast staining

the waxy cell makes the smear more likely to wash off during rinsing

how long is acid alchol applied

5-10 seconds

why is the slide steamed during acid fast staining

heat helps carbolfuchsin penetrate the waxy cell wall.

penicillin

an antibiotic produced by penicillium chrysogenum that prevents the cross-linking of peptidoglycans, which interferes with the final stages of bacterial cell wall synthesis

mordant

a substance used to intensify a stain by increasing its affinity for the specimen or forming a complex with the dye.

• helps dye remain attached to gram positive cells

synergism of antimicrobial drugs

the effect of two or more antimicrobial drugs used simultaneously being more effective than when used alone.

zone of inhibition

the clear area around an antimicrobial disk where bacterial growth has been inhibited.

• the size of the zone helps determine whether the bacteria are sensitive, intermediate, or resistant to the drug

teichoic acid

a polysaccharide located in the peptidoglycan layer of the cell walls of gram positive bacteria that regulates cation movement, is involved in cell growth, and provides antigenic specificity.

peptidoglycan

a carbohydrate and protein complex found in bacterial cell walls that helps protect the cell.

• gram positive: thick layer

• gram negative: thin layer