LAB QUIZ 4: SIMPLE STAIN + SMEAR PREP & NEGATIVE STAINING + GRAM STAINING

How do cells appear in a negative stain?

Cells appear transparent against a dark background

Iodine is a _______ that combines with the crystal violet and forms an _________ in gram _____ cells

Iodine is a MORDANT that combines with the crystal violet and forms an INSOLUBLE COMPLEX in gram POSITIVE cells

What happens when you try to counterstain a G POS cell?

Nothing. It's still purple because crystal violet is more intense of a stain than Safranin

What is the morphology of cocci?

Spherical

Negative stains are ________

acidic

Treponema pallidium morphology

causative agent of syphillis, is a SPIROCHAETE

How can you identify Corynebacterium diphtheriae?

internal structures like polyphosphate granules (volutin or metachromatic granules)

What is the 1st step in a smear prep for a SOLD medium?

place 1 or 2 loopfuls of water on the slide and then with an inoculating loop disperse the organism in the water.

What is the 1st step in a smear prep for a LIQUID medium?

place two or more loopfuls of the liquid medium directly on the slide

What is the definition of Simple Staining?

- the use of a single stain to color a bacterial cell

Why do we heat fix cell smears?

A smear of cells is heat fixed so it can adhere the cells to the slide and preserve structural integrity

What do you do after adding a primary stain to a gram stain?

Add Gram's Iodine

What dye is repelled by bacterial cells and stains the background of a slide?

An acidic dye

What is the name of the microorganism used in lab?

Bacillus coreus & corynebacterium diphtheriae

GRAM NEG cells have a cell wall that consists of an ______ _________ that covers a much thinner layer of peptidoglycan

GRAM NEG cells have a cell wall that consists of an outer membrane that covers a much thinner layer of peptidoglycan

What color is Gram NEGATIVE?

Gram NEGATIVE is decolorized and must be counterstained by a red dye

What are examples of negative stain?

India ink and nigrosin

What is the definition of SIMPLE STAINING?

Simple staining is a single stain to color/stain organism ex: crystal violet, methylene blue, basic fushin

Define Metachromatic granules

distinct reddish-purple granules within cells that show up when the organisms are stained with methylene blue ex: a polymetaphosphate such as VOLUTIN

What are the common dyes for simple staining?

methylene blue, basic fuchsin and crystal violet they are basic dyes

Negative stains are useful for:

studying the morphology of bacterial cells and characterizing some of the external structures that are associated with bacterial cells

What are the 3 goals of preparing a smear?

1. making sure the cells adhere to the slide 2. Making sure that the cells do not shrink during staining 3. Prep thin smears because it depends on how you can see individual cells and their arrangement/details

There are 2 methods for negative staining. What are they?

1. organisms are mixed in a DROP of NIGROSIN and SPREAD over the slide WITH ANOTHER SLIDE. -the goal is to produce a smear that is THICK AT ONE END and FEATHER THIN at the OTHER END 2. organisms are mixed in only a LOOPFUL of nigrosin insrtead of a full drop. organisms are spread over a smaller area in the center of the slide with an inocculating needle. NO SPREAD

What are the characteristics of SMEAR PREP?

1. really thin 2. durable 3. accurate determination of size

Why do we heat fix?

1. to stick cells to their slide 2. kill cells

Why are some bacteria considered gram-variable?

Because some cells will retain the crystal violet stain while others will not and appear red from the counterstain

What types of Gram stains are there?

Gram POSITIVE and Gram NEGATIVE

What color is Gram POSITIVE?

Gram POSITIVE retains a purple dye

GRAM POS cells have a thick layer of _______ that compromises the _______ _______ of organisms

Gram positive cells have a thick layer of PEPTIDOGLYCAN that compromises the CELL WALL of organisms

What is morphology?

Morphology is the shape of cells

Do you heat fix negative stains?

NO. "For negative stains cells are NOT USUALLY HET FIXED prior to the app;ication of the negative stains"

Negative stains have a _________ charged __________ that does not penetrate the cell but rather is ________ by the similarly _______ ______ _________.

Negative stains have a NEGATIVELY charged CHROMOPHORE that does not penetrate the cell but rather is REPELLED by the similarly CHARGED BACTERIAL CELL.

What do you stain both G POS and G NEG bacteria?

Primary stain like crystal violet

What do you use to counterstain a gram Negative cell?

Safranin. It makes the G NEG cell pink or red

Why do we stain cell smears?

Smears are stained with various dyes to enhance cell features and structures

What is the morphology of pathogens?

Spiral/helical

which organism can you view well with negative staining?

Spirochetes because they are very thin cells

What are the 2 organisms used in Gram Staining lab? What colors should they appear?

Staphylococcus aureus (purple cocci) & Pseudomonas aeruginosa(pink/red rods) & Bacillus megaterium (transparent)

How do you know when a bacteria is Gram POSITIVE?

When you add a decolorization such as alcohol or acetone and the crystal violet iodine complex is retained. It is still purple

How do you know when a bacteria is Gram NEGATIVE?

When you add a decolorization such as alcohol or acetone and the dye-mordant complex is removed, leaving them COLORLESS

How does negative staining look when you combine positive staining? Why?

You do this to better demonstrate capsules and sturctures. The capsule will have a halo surrounding the positively stained cell against a dark background

What is a basic dye?

a basic dye has color-bearing ionic groups (chromophores) that are positively charged (cationic) and work well with bacterial cells that have chemical groups on their surfaces that confer a net negative charged to the cell. ex: methylene blue, basic fuchsin and crystal violet.

What's another thing that negative staining is useful for?

accurately determining cell dimensions bc cells dont shrink because there's no heat fixation

With what do you decolorize cells with?

alcohol or acetone

What is an acidic dye?

an anionic chromophore (negative charged dye) -these stained are repelled by bacterial cells and stain the background of the slide

The _______ of a surrounding cell is colored by the negative stain

background

What is acid-fast bacteria?

bacteria with cell walls made of "waxy" lipids ex: mycobacteria

Vibrio cholerae morphology

bacterium responsible for cholera, COMMA SHAPED bacterium

What is mycobacteria?

causative agents of tuberculosis and Hansen's disease. An example of acid-fast bacteria

What is the Gram stain? When Hans Gran discovered it?

certain stains were retained by some types of bacterial cells but removed from others during the staining process

The Gram stain is an example of _______

differential stain

how can you collect organisms for negative staining?

from oral organisms (between your teeth) and from cultures

What is the procedure for a negative stain?

mixing the organism with a small amount of stain and spreading a very thin film over the surface of the slide

Define Palisade arrangement

pertains to parallel arrangement of rod-shaped cells. "picket fence" ex: common to corynebacteria

Define Pleomorphism

pertains to the irregularity of form; demonstration of several different shapes ex: C. diphtheriae : rod shaped but also seems spermlike

What are some characteristics to identify bacteria using corynebacteria stain?

pleomorphism, metachromatic granules, and palisade arrangement of cells

After adding iodine to gram stains, what is the color?

purple

What is the morphology of bacillus?

rods

What is a cationic chromophore?

see "basic dye'

cultures used in lab for simple staining

staphylococcus aureus streptococcus lactis bacillus megaterium corynebacterium xerosis

Why do we use gram staining?

to identify new unknown bacteria