Exercise 5: Preparation of Smears and Simple Staining

Preparing smears:

-start with a clean slide -handle slides by the edge mark bottom of the slide with a circle to mark where bacteria goes -sterilize your inoculating loop heating to redness allow the loop to cool without touching it or setting it down about 30 seconds -from solid media- put a drop of water in the circle -from liquid media-place 2 or 3 loopfuls of the liquid culture on the slide with the sterile loop -allow the smears to dry at room temp

The dry smear is passed through a ____________ several times to __________ the bacteria.

Bunsen burner heat fix

Methylene blue chloride

Methylene blue+ + Cl- (Chromophore)

Who introduced into microbiology the procedure of air drying, chemical fixation and aniline dyes?

Robert Koch

Methylene blue can be prepared as a basic stain or an acidic stain. How much would the pH of the stain affect the staining of bacteria?

The pH will determine if the bacteria will have a particular charge

What value is a simple stain?

a simple stain enhances the contrast between the bacterium and its surrounding material and permits greater clarity of detail. Simple stains can be used to determine cell morphology size and arrangement

Define a smear

a thin film of bacterial cells put on a slide

if it is a negative ion it is an ______________.

acidic stain

Fixing denatures bacterial enzymes preventing them from digesting cell parts which cause the cells to break is a process called _____________?

autolysis

What microbe is this?

bacillus subtilis

Staining bacteria enhances the _______ between and the surrounding environment and permits observation of greater detail and resolution than wet mounts do.

bacteria

A smear is prepared by:

bacterial broth- using an inoculating loop take a small of amount of the liquid and smear it on to the slide and let completely air dry solid-remove a small amount of bacterial culture mix with a drop of water on the slide and allow to air dry.

If the chromophore is a positive ion like the previous Methylene blue equation (Methylene blue+)the stain is considered a ______________.

basic stain

Another method of fixing smear is _____________?

chemically fixing

The ion that is colored is referred to as a _____________.

chromophore

Multiple reagents being used is called______________ and the bacteria react to the reagents the same or different?

differential stains different (differential stains)

Bacteria can be seen without staining. Why is Koch's recommendation for fixing and staining important for microbiology?

fixing and staining allows the bacteria to be saved and re-examined and shared for study

Name the various tools you can use to transfer bacteria to a microscope slide.

inoculating loop and a needle

What is the purpose of heating fixing the smear?

kill the bacteria and preserves them with minimal shrinkage and damage to the microbe.

To chemically-fix the bacteria cover the smear with 95% _____________ for 1 minute.

methanol

Fixing also preserves _________ with minimal shrinkage or distortion when stained.

microbes

Coagulated ________ from the cells will cause cells to stick to the slide

proteins

The dyes are usually _______ but some are _____ or ________.

salts acids or bases composed of charged colored ions

With simple techniques may involve _______________ in which only _______ ________ is used and all the bacteria are similarly stained.

simple stains one reagent

How are microorganisms prepared for staining?

smearing them onto a microscope slide

What microbe is this?

staphylococcus aureus

Most stains used in microbiology are?

synthetic aniline dyes from benzene

Why are structural stains used?

to identify specific parts of microorganisms

Can dyes other than methylene blue be used for direct staining?

yes, crystal violet, basic fuchsin and safranin are all dyes that can be used in direct staining because they are cationic with a positive charge

How does alcohol chemically fix the bacteria?

•It removes water, it dehydrates the cells. This is important when mounting the cells in non-aqueous mounting medium. •It denatures proteins. This way the metabolism of the cell is stopped and the cell dies. The metabolism is dependent on enzymes, which are proteins. •It dissolves and removes lipids. The cell membrane(s) of the bacteria is harmed by the alcohol.