Negative And Simple Staining

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The most important feature of negative staining is that

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no heat is applied

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Why is it most important to ensure no heat is applied in negative staining

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so there is no possibility of cell distortion as a result of overheating and true morphology is seen.

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Explain the concept of negative staining

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acidic stains (dyes having negatively charged dye molecules) are repelled from the negatively charged surface of a bacterium. Therefore bacteria appear clear in a dark background.

Front 4

Examples of stains or dyes used for negative stains include

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nigrosin, eosin and India ink.

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First step in preparing a negative stain is to add a drop of ________ to one end of a clean slide

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nigrosin

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Second in preparing a negative stain is to mix a loopful of bacteria with the drop of ________ on the slide

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nigrosine

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Third in negative staining is to use another slide to

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smear the nigrosin/bacteria mixture across the surface of the slide and air dry

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Negative stains should be viewed with microscope how

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the slide is observed under oil immersion

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In a negative stain, the bacteria are seen as

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bright areas in a dark background.

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Negative Staining is not a suitable technique for viewing

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capsules.

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All the staining techniques performed in this lab involve preparation of a bacterial smear and heat fixing with the exception of

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negative and capsule staining

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In preparing a smear, the tendency of all beginning students is to add too much inoculum.

As a general rule for preparing smears, if the culture you are using is liquid, you do not need to add

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water to the slide.

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As a general rule for preparing smears, if the culture is growing on agar, you must add

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water to the slide first.

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When preparing a smear, mix the organisms into the water with inoculating loop so an even

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emulsion is formed

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When preparing a smear, it is important that you do not any clumps of _____ on the slides

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Agar

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Your bacterial smear should be the consistency of

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very diluted milk.

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Bacterial smears only need to be about the diameter of

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a pencil eraser. In fact many smears can be placed on a single slide.

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For every staining procedure involving a smear, what must be ensured of the bacterial smear prior to heat fixing

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the bacterial smear must be completely dry prior to heat fixing.

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It is not acceptable to speed up drying by

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blowing, using the Bunsen burner, waving the slide in the air.

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Why is it not acceptable to assist in the drying of bacterial smears

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Risk of creating aerosols

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What is the acceptable way to aid bacterial smears in drying

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slide warmers located in the lab

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What may be used for heat fixing instead of Bunsen Burners

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Slide warmers at high temp

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Beginning microbiology students are encouraged to dry bacterial smears how

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allow slides to air dry rather than using the slide warmers.

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If you use small loops of water to generate your smears, they usually dry in

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less than one minute

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A critical and necessary step for a good staining procedure is

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Heat Fixing

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Heating fixing does what

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fixes bacteria to the glass so the slide is permanent, kills cells and inactivates enzymes within the cells which can cause cells to rupture or degrade

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Too much heat fixing can cause

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distortion and breakage of cells.

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Once cells are heat fixed, they may be

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stored prior to staining.

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Once a bacterial smear is heat fixed, it is ready for

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staining.

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A variety of stains can be used in a simple stain as long as they are

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basic or positively charged.

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Bacterial cells have __________ charged surfaces

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negatively

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In simple staining procedures, positively charged dye molecules will be

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attracted to the negatively charged bacterial cells

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Positively charged stains include

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crystal violet, safranin, and methylene blue.

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This substance is also a basic stain; however, it also contains phenol (carbolic acid) and is more suitable for specialized stains such as the acid-fast technique.

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Carbolfuchsin

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Carbolfuchsin is also a basic stain but due to its phenol content it is more suited for

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acid fast staining

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Once the staining procedure is finished, residual dye is

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washed away

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After washing away residual stain in a simple staining the slide is then

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blotted dry.

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The cells retain the stain, and the background should be

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clear.

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Are cover slides required for simple staining

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No cover slip is required for these slides

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With simple staining oil is added

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directly to the slide for observation

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Stained and heat fixed slides may be

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stored and observed later

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A negative stain is uses a stain which repels from

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bacterial cell walls

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Using a stain which repels from cell walls will

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stain the background and bacteria will not be stained. Causes a color background with bright and clear cells

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A simple stain uses a dye which is attracted to

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bacterial cell walls

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Using a stain which is attracted to cell walls will

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color bacteria so that they appear as colored shapes in a clear background.

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These simple staining techniques work the same for all bacteria regardless of

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cell wall composition.

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This staining technique is powerful because it does not require heat fixing. This eliminates cell distortion and shows true cell morphology

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Negative staining

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Bacillus subtilis

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a large bacillus which produces endospores.

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Term) Endospores

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resistant, dormant structures which are produced by members of the Bacillus and Clostridium genus as a means of surviving harsh environments.

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These dormant structures which are produced by the Bacillus and Clostridium genus are resistant to simple staining procedures.

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Endospores

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Micrococcus luteus

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a coccus which occurs in tetrads.

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Corynebacterium pseudodiphtheriticum

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a nonsporing bacillus which forms club or cigar shapes

Front 53

Bug collection slants will be saved and labeled how

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label with each person’s last name, table number and lab section

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Bug collection slants will be placed in a can at what temp

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25C.

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You will use your bug collection when needed to

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set up experiments

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If a bug collection culture is contaminated, you must

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re-isolate the organism on a TSA streak plate and reestablish a working slant.

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If you run out of a bug collection culture you must

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make a new slant from cells left on the old one.

Front 58

Steps in a simple stain

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1. Generate bacterial smear
2. Air Dry
3. Heat Fix
4. Add stain for 1 minute
5. Wash with distilled water
6. Blot dry with towel
7. Wash back of slide with alcohol
8. View with oil immersion.
Note: Oil is added directly on top of smear

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How to generate smear

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mix loopful of water with bacterial sample

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The purpose of heat fixing is to

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permanently attach bacterial cells to the glass slide and allow storage of preserved cultures for long periods of time.

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Heat fixing also inactivates enzymes within the bacterial cells which, if active, can

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degrade the cell wall over time and destroy the sample

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Methylene blue is a basic dye (positively charged) which is

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attracted to the negatively charged bacterial cell wall

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After simple staining, wash back of slide with

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alcohol

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Steps in a Negative stain

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1. Add 1 drop os nigrosin to clean slide
2. Mix in bacteria
3. Use a clean slide at 45 degree angle to smear dye and bacteria across slide
4. Air Dry
5. Observe using oil immersion
Note: Put oil directly on top of dried sample

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Using the simple stain, B. subtilis will appear as

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blue bacilli with endospores

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Endospores will appear how following staining

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Endospores will look like clear grains of rice.

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Using simple staining M. luteus will appear

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blue cocci arranged in tetrads, diplococci, staphylococci and streptococci

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Using simple staining C. pseudodiphtheriticum will appear

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blue bacilli which are pleomorphic. Look for “P” or “9" shapes as well as cigar or club shapes

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C. psuedodiptheriticum is pleomorphic which means it may have

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more than one shape.

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Using the negative stain, you will see the true

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morphology of microbes.

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Using negative staining, B. subtilis and C. pseudodiphtheriticum will appear

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as bright bacilli in a purple background.

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Using negative staining M. luteus will appear as

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clusters of bright cocci in a dark purple background.

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Pic) Simple stain of bacillus

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Pic) Simple stain of Micrococcus

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Pic) Negative stain of bacillus

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Pic) Negative stain of micrococcus

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