Microbiology Lab exam 1 - Smears and Simple Staining

Describe the potential consequences of making a smear that is too thick?

- does not allow for proper penetration of light through smear - bacterial cells are often packed too closely together

For preparation of a smear on a slide, what is the purpose of the heat fixation? What problems can arise when the slide is heated in a flame?

-Heat fixation of smears kills the bacterial cells and causes them to adhere to the glass so that they do not get washed off during staining -overheating can damage and dehydrate the cells causing them to distort in shape - the glass could crack or shatter if heated too long

fusiform bacteria has what shape?

-they are rods that are wider in the middle and taper off on the ends; -prevalent in the human mouth.. not the same as bacilli

How to screw up a smear?

1) Forget to clean the slide 2) use too much material ( suspension should be just barely cloudy) 3) use so much liquid that it takes forever to dry 4) heat the smear before letting it air dry, boiling the bacteria instead of attaching them 5) Overheat the smear, melting cell walls and possibly breaking the slide

What are you able to determine by doing a "good" smear?

1) the morphology of cells (rods, cocci, or other bacterial shapes) 2) the arrangement of cells (such as single cells, chains or clusters) 3) internal structures, such as endospores and cell inclusions

What are the goals in preparing a smear (3)

1) to adhere the cells to the microscope slide(without overheating them) so they're not washed off during staining and washing procedures 2)It is important to ensure that shrinkage of cells does not occur during the staining 3) to prepare thin smears because the thickness of a smear will determine if you can visualize individual cells, their arrangement, or details regarding microstructures associated with cells

Describe the steps of doing a simple stain (3)

1. A bacterial smear is stained with methylene blue for 1 min. (this is done on heat fixed smears(slides) 2. Stain is briefly washed off slide with water 3. Water drops are carefully blotted off slide with bibulous paper.

What are some characteristics of Cornebacteria? (3)

1. Pleomorphism 2. Metachromatic 3. Palisade arrangement

Steps to making smears from plates-----> slants

1. Two loopfuls of water are placed in center of "target circle" 2. A very small smount of organism is dispersed with inoculating loop in water over entire area of center of slide. (disperse it pretty wide (bigger circle). 3. The smear is allowed to dry at room temp 4. After smear has completely dried, place the slide on the incinerator (make sure it's the bottom side of slide) and pass it over the top of the incinerator for 15 seconds.

Steps to making smears from broth cultures

1. obviously shake the culture vigorously and transfer two loopfuls of organism to the center of the slide. 2. Organisms are dispersed over entire area in the center of slide 3. The smear is allowed to dry at room temperature 4. After smear has completely dried, place the slide on the incinerator (make sure it's the bottom side) and pass it over the top of the incinerator for 15 seconds.

What is meant by palisade arrangement of cells?

A parallel arrangement of rod shaped cells characteristic of the Corynebacteria

What is the importance of making a smear?

A smear allows us to stain and rinse slides without washing off the bacterial cells

What is the difference between basic and acidic dyes?

Basic dyes are positively charged(cations) and therefore are attracted to negatively(anion) charged bacterial cells; acidic dyes are negatively repelled by negatively charged bacterial cells.(they are both anions in this case)

Why is there little contrast in a bacterial cell between the actual cell and it's surrounding aqueous environment?

Because bacterial cells are composed of approximately 80% water, there is little contrast between the cell and the surrounding aqueous environment in which they occur.

Why do bacterial cells work so well with the basic dyes?

Because the bacterial cells themselves are negatively charged (anion) and the basic dyes are positively charged (cations) There is a pronounced electrostatic attraction between the cell(negatively charged) and the cationic(positively charged) chromosphore of the stain, and the result is stained cells.

Cocci can occur how? Also, what shape is Cocci?

Cocci(plural) can occur single, in chains, in tetrads, or in irregular clusters; Cocci is circular in shape

what is the name of the bacteria that has a round & rod appearance?

Coccobacillus

Which aseptic techniques are involved in making smears?

Flaming the inoculating loop or needle and also heating the mouth of the tubes if you are using them for transfering.. obviously if you are transfering from a nutrient agar dish then you would not heat that for sterilization.

What is the purpose of heat-fixing a smear?

Heat fixation is a technique used in organism staining that is able to kill organisms, adhere them to the slides being used, and alter them so they can take on the stains being used.

After making a smear, what must you do to the inoculating needle or loop before putting it down on the table?

Heat it with incinerator to sterilize it

What is the most difficult concept for students to understand about making slides from solid media?

It only takes a small amount of material from the solid media to make a smear

What does streptococci look like?

It's a chain of spherical bacteria

What does Staphlococci look like?

It's grape looking clusters of spherical bacteria

What is the problem with a smear that is too thin? What is the problem with a smear that is too thick?

It's important to prepare thin smears, but obviously enough so you can actually find them on the microscope. Too thick of smears with large clumps can obscure details about arrangement and the presence of internal structures. Also, stain can become entrapped in the clumps of cells preventing removal by destaining and washing and leading to erroneous results for staining reactions.

are distinct reddish purple granules within cells that show up when the organisms are stained with methylane blue. These granules are masses of volutin, a polymetaphosphate

Metachromatic granules

What dyes are commonly used for performing simple staining?

Methylene blue(we used this one) or basic fuchsin or crystal violet

Pertains to parallel arrangement of rod shaped cells. Characteristic that is called "picket fence" arrangement

Palisade arrangement (side by side long ways)

pertains to irregularity of form - that is demonstrating several different shapes

Pleomorphism

If you were working with an unlabeled simple stained smear, would you be bale to identify the bacterial species by observing the slide under the microscope? Why or why not?

Simple staining only indicates size, shape & arrangement of species. Many different species have similar characteristics, therefore this would not be enough to determine species.

What can be used to determine the morphology of bacterial cells and arrangements?

Simple stains

When are smears ready to heat-fix?

Smears are ready to heat fix when they are completely air-dried.

Before collecting a sample, why is it necessary to touch the flamed inoculating needle to an area of plain agar first?

So the needle or loop will be cooled enough (in a sterile way) before making contact with the bacterial cells.

What is the difference between spirilla and spirochete?

Spirilla are spiral prokaryotes that are relatively short and rigid, while spirochete are spiral prokaryotes that contain longer and more flexible cells which cause Lyme disease and syphilis.

When making a smear from an agar colony, what is the advantage of using a small drop of water to spread the cells?

The smaller the drop, the faster it dries

Why is it important to do a simple stain with Cornebacteria ?

The stain will demonstrate unique characteristics of these bacteria that are used in their identification.

What do tetrad arrangement in bacteria look like?

They are in groups of 4's

What do bacteria on solid media tend to do?

They tend to cling to each other and must be dispersed sufficiently by dilution in water- unless this is done, the smear will be too thick

What is the disadvantage of making a thick smear?

Thick smears of cells with large clumps can obscure details about arrangement and the presence of internal structures(such as granules, etc)

What does simple staining refer to?

To use a single stain to color a bacterial cell.

Crystal violet is an example of what type of stain?

a basic stain which has a positive(cation) charge

If you are preparing a smear from solid media what must be added to the slide before adding the cells?

a loop-ful(or couple) of water

How can you enhance the contrast of bacterial cells so they can be visualized with a brightfield microscope?

a smear of cells is prepared on a microscope slide and usually heat fixed to adhere the cells to the slide, and importantly, to preserve their structural integrity. Smears are often stained using various dyes to enhance cell features and structures.

What type of dyes have anionic(negatively charged) chromosphores?

acidic dyes

What type of stains are repelled by bacterial cells, and will stain the background instead?

acidic dyes - because they are anionic(negative charge), and bacterial cells also have a anionic charge(negative charge), they repell each other, and the dye will stain the background of the slide instead.

cells that have rounded, flat, or tapered ends

bacilli

What 3 common types of shapes(of the bacteria), can be determined with the simple stain?

bacilli(rods), cocci(spherical), and spiral(corkscrew)

What are Methylene blue, basic fuchsin, and crystal violet referred to?

basic dyes- because they have color bearing ionic groups (chromospores) that are positively charged (cationic) They work well with bacterial cells that have negative charge (anionic)

How does smear preparation of cells from a liquid medium differ from preparation of cells from a solid medium?

cells from a solid medium are placed in a drop of water on the slide, then smeared ; from a loopful of culture from a liquid culture, placing a drop of water is not required.

Describe this cell shape and arrangement (Streptococci)

circular and in chains

Describe this cell shape and arrangement ( Staphylococci)

circular in clusters

What are chromosphores?

color bearing ions

What are some common types of Spherical(cocci)

diplococci and tetrads, streptococci, and staphlococci

Why is it important to limit the quantity of cells used to prepare a smear?

if too many cells are used to create a smear, it will be difficult to distinguish individual cell shapes and arrangements from large clumps of cells. Also staining and destaining techniques don't work on clumped cells

What is the first step in preparing a bacteriological smear?

it differs according to the source of the organism If the bacteria are growing in a liquid medium(broths, milk, saliva, urine, etc), one starts by placing two or more loopfuls of the liquid medium directly on the slide From a solid media such as nutrient agar, blood agar, or some part of the body, one starts by placing one or two loopfuls of water on the slide and then using an inoculating loop to disperse the organisms in water.