Staining methods

Simple, Negative, Capsular, Gram, Spore, Acid-fast, and Motility

Identify the different types of staining methods we have done in class thus far

Put 5 loops of broth on slide (being sure to flame loop between loops and the top of the tube prior inoculating and closing with lid) let completely air dry then heat fix

Identify the steps for preparing a liqiud smear prep

Place 2 -3 loops of sterile water on the slide first. Then emulsify one needle of the organism from the solid. Allow to completely air dry and heat fix.

Identify the steps for preparing a solid smear

Cover both slides with stain (methylene blue), let sit for about a minute, rinse with deionized water, blot dry with bibulous paper

Identify the steps to take after the bacterial smear prep for both simple staining's completed in lab.

Spread Method. Place Acid (India Ink) towards one end of slide, inoculate bacteria into India Ink, take second slide at an angle and slide it into the mixture just until it comes into contact, then drag the slide back across to spread the mixture across the slide, let air-dry and NO HEAT FIXING, observe under oil immersion lens.

What kind of method did we do for the Negative Stain and describe procedure

Acid stains are anionic, meaning they have a negative charge. With bacteria having a negative charge, a (-) and a (-) will repel on another causing the background to stain and the bacteria staying translucent

What occurs if you use an acid for staining and why

methylene blue

What type of dye did we use in class for the simple stain

No, because the stain is liquid itself

Do you need sterile water for Negative Staining Method and why?

no because it's already a liquid

Do you need to use water with a broth?

False; because the stain does not penetrate the microorganism, instead it obliterates the background leaving the organism transparent and visible in a darkened field

Negative staining is a bacterial staining method, true or false and why?

nigrosin, eosin, and Congo red

What are examples of an acidic dye?

Kills bacteria, makes them adhere to the slide, coagulates cytoplasmic protiens to make them more visible.

What is the purpose for heat staining?

determines cell morphology, size and arrangement

What is the simple staining technique useful for?

Smear preparation

What is the starting point for all slide preparations?

India Ink

What type of stain did we use in class for the Negative Staining Method?

Negative staining

What type of staining uses an acidic stain and uses no heat fixing for the smear prep?

If after completing a simple stain and observing the specimen and it looked distorted or undefinable, more than likely the cell wall was too delicate to withstand the heat fixing.

When would you or what would prompt you to do a Negative Stain Method

because these bacteria are too delicate to withstand heat-fixing and distorts specimen. Also when accurate size is crucial, and produces minimal shrinkage.

Why do you not use heat fixing for Negative Staining

due to the bacteria having a slight negative charge and the chromophores having a + charge creating the attraction.

Why is chromophores good to use for staining?

to prevent contamination

Why must you flame your loop between

Useful for determining cell arrangement, morphology, and size. Also useful for studying spirochetes that don't stain readily with ordinary dyes

Why would we use the Negative Stain Method?

emulsion

another word for emulsion

chromophores

color bearing ions

methylene blue, crystal violet, basic fuchsin, safranin

common stains used for simple stains

+ charge; methylene blue, crystal violet, basic fuchsin, safranin

what type of charge is a chromophore and give examples