Lab: Simple Staining

Positive staining

Bacterial cells have a net negative charge, so they can be stained by a positive chromophore such as Methylene+ chloride-. This is also referred to as a basic dye because the chromophore is (+) charged. The methylene contains the (+) charged color bearing ion so will stick to the negative cell, staining it. We will use a colony from the first lab (Where in the world are Microorganisms) to do a simple stain. Choose a colony from your plates you want to work with. We will stain that. Most staining procedures require a preparation of a smear of bacterial. The smear prep is important in 1) ensuring that the microorganisms are attached to the slide and will not wash off during the staining procedure and 2) the cells are killed in the process. Heat Fixation Procedure: Staining with Methylene Blue: Additional notes on Simple Stain

Bacterial cells are made up of about

80% water, so there is very little contrast between the cells and the background which make them very difficult to see. However, to determine the morphology of the cells one must be able to visualize them using brightfield microscopy.

What are the advantages and disadvantages of Positive stain?

Adv: are heat fixing so not dealing with a live bacterial cell Dis: are heat fixing so the bacterial cell may have a bit of a distortion to it and not be the more natural shape

What are the advantages and disadvantages of the negative stain?

Adv: do not have to heat fix because the heat distorts the cell a little bit so when you do not heat you do not distort it so will have more of the natural appearance Dis: do not have to heat fix but dealing with a live bacterial cell

Procedure: (Negative Staining)

For this technique, you will need two slides. Place a drop of Nigrosin / India Ink on a slide. With a toothpick, collect some plaque from between your teeth with a toothpick and disperse the plaque into the drop of Nigrosin/India Ink. Using a second slide spread the drop of Nigrosin and plaque across the slide as shown in the figure below. Let the slide dry. Note: you have not heat fixed this slide, so the bacteria are still living and will have a more natural shape and size. The disadvantage to this method is that the bacteria are still living and handled with care.

What step normally associated with staining bacterial cell is omitted when the dimensions of cells are determined? Why?

Heat fixation is normally omitted when determining dimensions of bacterial cells because heat will cause cells to shrink.

When staining

do not wear good clothes because you can get the methylene blue on you

Coccobacillus / coccobacilli

elongated coccus; short, thick, oval-shaped bacterial rod

What type of chromophore is associated with a negative stain.

eosinate

Generally use

methylene blue = methylene chloride. Methylene = color bearing ion which calls the chromophore; + charge. Chloride = negatively charged

The bacteria are

negatively charged. As a result, we are going to use a stain that is positively charged.

With the - stain

that is how you do it. Put a drop of nigrosin/India ink on the slide and ours come in a drop bottle so do not have to do it with an inoculating loop. Then take your sample and number of ways you can do this: more interesting = can take plaque from your teeth and scrape between your teeth with a toothpick and put your sample within the drop and then take a second slide and spread. Spread the nigrosin across the slide. Do not want it to be thick or too thin and spread it out so you can look at the slide Thin layer can still see but too thin cannot and thicker layer can see but difficult and sometimes if too thick cannot hear it Warning: put the slides in the brown waste cans on the table not in the cylinders on the table once done and throw it in the brown waste cans on the table

When a single stain is used

the staining process is referred to as simple staining. These staining procedures are useful in determine the morphology (shape and arrangement) of the bacterial cell. There are two very basic staining procedures that will allow you to visualize the shape and arrangement of bacterial cells, positive and negative staining. These staining procedures depend on the use chromophores (color-bearing ions) to either stain the cells or to stain the background

If the objects you are looking at are very bright

they are probably just cracks in the dried ink/nigrosin.

Steps: (Positive stain)

1. Draw a circle on the slide with a grease marking pencil/sharpie. Does not matter which but will notice because then will put a drop of water on the slide. If you use the grease marking pencil, that may come off. Can always put it back on once done with the staining process 2. When you are done, you need to heat fix your sample. 3. Take the methylene blue and put it on the slide where the sample is and then wash it off 4. Blot it dry 5. When you are done, you will look at it under the microscope. Will dry and blot it off and put it under that. Remember what did last week with the microscope: start with scanning, then low, then high, and then oil like last week

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

1. It will help attach the cells to the slide so they do not wash off and 2. They will kill the cells. Bacterial cell may have a bit of distortion to it and not be the more natural shape

What the heat fixing does is: (Steps: - Positive stain)

1. It will help attach the cells to the slide so they do not wash off and 2. They will kill the cells. This is beneficial if you are dealing with a pathogen and you do not want it to be living when you are working with it. That is one of the advantages to this particular method. Put a circle on then a drop of water and then heat fix. Heat things slowly because you do not want to burn it and do not want to burn the cells and sear them. Will know this. Sometimes be yellow or brownish-color, there is a particular broth sometimes used that causes a nice yellow color and not burning it. Will smell it if you burn it and will know. Slowly heat fix

The purpose of the circle: (Steps: - Positive stain)

1. Know where you put your sample in case you are using a sample that does not have a lot of cells in it, which is not going to be the problem for us. 2. So that if you cannot find anything, you have something on the slide that you can go and focus on and then move back If you put the circle on the same side of the slide that you put your sample, they are in the same field of view so you can always go back and find if you focus on the grease mark and find your sample if they are in the same field of view. If that grease mark washes off, when you are done with the staining process, you can always put another mark on the slide so you know so it does not matter

Steps of the Procedure: (Negative Staining)

1. Organisms are dispersed into a small drop of nigrosin/india ink. Drop should not exceed 1/8" diameter and should be near the end of the slide 2. Spreader slide is moved toward drop of suspension until it contacts the drop, causing the liquid to spread along its spreading edge 3. Once the spreader slide contacts the drop on the bottom slide, the suspension will spread out along the spreading edge as shown 4. Spreader side is pushed to the left, dragging the suspension over the bottom slide. After the slide is air-dried, it may be examined under oil immersion

Steps of + staining

1. Take colony from the plate. First thing do when do a simple stain is draw a grease mark on slide in a circle so know where putting it 2 reasons: know where to put the sample and if need to and cannot find it under the microscopy power, can focus on the grease mark. That is why I put it on the side. Some things online say differently 2. Put a drop of water on the slide. 3. Take a needle and sterilize the needle with fire. Not the handle because not going into a test tube. Just need the loop. 4. Wait for the needle to cool for about 1 minute. 5. Open the agar plate and go in and poke that colony. Do not need a lot. Get a little on the loop 5. Break that off inside the drop of water. Break up the clump 6. Sterilize needle again to get off what might have been on there and put it down 7. Now I need to heat fix it. See online says to let it air dry. Reasons tell you that because they do that is because the heat can distort and break the bacteria open, but what is found is that it takes it too long to evaporate. What to do is to heat fix it while evaporating the water. Have to do it slowly. Pass it through the flame slowly, not fast. Three times and pause. Do not want to get it too hot. Wait about 3 seconds then start again. Hold the slide with clamps While heat fixing doing two things: killing the bacteria that might be on the slide and attaching it to the slide so it will not wash off in the staining procedure 8. Keep passing it through the flame until all of the water evaporates. Will take a little bit. 9. Wait for your slide to cool and do not put it on the cool table because it might break 10. Set it on the plate with the cooling rack and let it cool. Look for red mark and may wash off when wash it but you can always put it back on 11. Put enough methylene blue onto coat the smear and once done, let it sit for 30 sec-1 min and then wash it with water 12. Turn the burner off while waiting because it is not needed anymore 13. Tip the slide and rinse it with water on both the front and back 14. Clean it off by tapping it and rubbing it 15. Should see the bacteria and then do the microscopy part of it

From a plate or slant (Heat Fixation Procedure: - Positive staining)

Draw a target circle on the slide. Place a drop of water inside your target circle. You can use a loop to place a drop Obtain a very small of bacterial from a colony on your petri dish and disperse it in your water using the loop. Using a clothes pin pass the slide through the flame several times. You will want to do this very slowly (as demonstrated by your instructor). You do not want the water to boil. This will distort the shape of the bacterial cell. You want to slowly allow the water to evaporate from the slide Once all the water has evaporated from the slide, pass it through the flame a few more times to ensure the bacteria has been heat-fixed. Allow the slide to cool before continuing.

From Broth Culture (Heat Fixation Procedure: - Positive staining)

Draw a target circle on the slide. Using a sterile loop obtain a sample from a broth culture Disperse the loopful in the target circle and try to disperse it within the circle. Be sure the bubble on the loop has not popped before it hits the slide. You must see a drop of liquid on the slide. Using a clothes pin pass the slide through the flame several times. You will want to do this very slowly (as demonstrated by your instructor). You do not want the water to boil. This will distort the shape of the bacterial cell. You want to slowly allow the water to evaporate from the slide Once all the water has evaporated from the slide, pass it through the flame a few more times to ensure the bacteria has been heat-fixed. Allow the slide to cool before continuing.

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

Liquid medium: defined as water-based solutions that do not solidify at temps above freezing and that tend to flow freely when the container is tilted. These media, termed broths, milks, or infusions, are made by dissolving various solutes in distilled water. Growth occurs throughout the container and can present a dispersed/cloudy/flake appearance. Common lab medium = nutrient brother: contains beef extract and peptone dissolved in water. Other: methylene blue milk and litmus milk that contain whole milk and dyes. Fluid thioglycollate is slightly viscous brother used for determining patterns of growth in oxygen Solid medium: provides a firm surface on which cells can form discrete colonies and are advantageous for isolating and culturing bacteria and fungi. Two forms: liquefiable and nonliquefiable. liquefiable solid media, aka reversible solid media, contain a solidifying agent that changes its physical properties in response to temp. Far most widely used and effective of these agents is agar (polysaccharide isolated from the red alga of Gelidium; medium first employed by Dr. Hesse) are numerous. Solid at room temp and melts at the boiling temp of water. Once liquefied, agar does not resolidify until it cools to 42 degrees C so it can be inoculated and pouring in liquid form at temps that will not harm the microbes or the handler. Agar = flexible and moldable, and it provides a basic matrix to hold moisture and nutrients. Another useful property is that it is not readily digestible and thus not a nutrient for most microorganisms

Are pros and cons to both + and - stain

Most of the time people want to go with the + because it is easier to see

Negative Staining

Negative stains are useful in studying the morphology of bacterial cells and may be useful in identifying external structures such as capsules. The negative stain is acidic and therefore the chromophore is negatively charged. This means the chromophore will be repelled by the negatively charged bacteria and will adhere to the positively charged background. Examples of negatively charged stains include India ink, Nigrosin or Sodium+ eosinate-. This result of this procedure looks like a negative of a picture. Procedure:

What external bacterial cell structures can be demonstrated by a negative stain?

Negative stains can demonstrate capsules.

Staining with Methylene Blue: (Positive staining)

Now your slide is ready to be stained with Methylene chloride aka Methylene blue. Place the slide on a stain tray. Cover the smear with methylene chloride and allow the stain to sit for about a minute. Wash the stain off with water. Sometimes you will need to wash the bottom of the slide off as well. Be sure to remember which side of the slide has your specimen. Blot the slide with bibulous paper to dry it off. Find a cleaner sheet of paper in the pad for the blotting. Don't press too hard as you don't want to break the slide during the blotting process. Once the slide is dry you can view it on the microscope. If your target circle has washed off during the staining process, you can redraw the circle on the slide at this time. Sometimes the circle can serve as a guide if you cannot find the smear on the slide, as it is sometimes easier to find the grease mark. If your grease mark is in the same plane as your specimen, you will be able to slide over to your specimen after you have the grease mark in focus.

Negative stain:

Simple stain by negative stain Acidic stain and - chromophore/- charge to it. Instead of the basic dye, using a -. Similar charges repel each other so the - charge will be repelled by the - bacterium and it will not stain the bacterial cell. It will stain the background, not the bacterial cell. Like the - of a pic. Have the background dark and bacteria is light. Opposite of what expect - stains are India Ink, Nigrosin, and Na+ eosinate - (using the first two in ours). In this instance the chromophore (eosinate) is - charged and give you the opposite of the + stain because it will be repelled by the - bacterial cell In this instance, do not go up to oil. Will have a lot of black on that slide and do not want the objectives on the microscopes to get black because it will be difficult to clean. Darker background and lighter bacterial cell and can see multiple types of bacterial cells possible Adv: do not have to heat fix because the heat distorts the cell a little bit so when you do not heat you do not distort it so will have more of the natural appearance Dis: do not have to heat fix but dealing with a live bacterial cell

Heat Fixation Procedure: (Positive staining)

The first step in the smear prep depends on whether you are using a culture of bacteria from a broth tube or a petri dish.

Additional notes on Simple Stain (Positive staining)

The species Corynebacterium is commonly used to demonstrate a simple stain. Most bacteria are monomorphic (showing only one shape) but the species of Corynebacterium are pleomorphic, existing as more than one shape. It is essentially a rod-shaped organism, but, in older cultures the bacteria show a club shaped or sperm like appearance. Corynebacterium is also known for its palisade arrangement. This pertains to the arrangement of the cells, which exhibits more of a "picket fence" arrangement. Finally, in some simple stains of Corynebacterium, reddish purple granules appear within the cells. These are called metachromatic granules and consist of volutin, a polymetaphosphate and function as a storage site for this chemical.

Simple Stain Techniques:

Two ways to do a simple stain: + stain (Monday) and - stain (Wednesday)

What causes a stain to adhere to bacterial cells?

With a + simple stain, and theory behind it is, you are going to take a dye and in the + stain, the dye is + stained. It will bind to the bacterial cell which is - charge. Dye is + charged. What that means is the color-bearing ion, the part of the stain that has the color to it, is the chromophore (color-bearing ion) and it is +. So the + stain is going to attach to the negative bacterial cell and the bacterial cell will be stained.

Positive stain

With a + simple stain, and theory behind it is, you are going to take a dye and in the + stain, the dye is + stained. It will bind to the bacterial cell which is - charge. Dye is + charged. What that means is the color-bearing ion, the part of the stain that has the color to it, is the chromophore (color-bearing ion) and it is +. So the + stain is going to attach to the negative bacterial cell and the bacterial cell will be stained. Stain we use in the lab is methylene+ chloride-/methylene blue Methylene = color bearing ion and will bind to the bacterial cell and stain it blue Chloride has no color to it That is a simple stain: in the demo, is going be doing is taking the bacterial cells from that plates that did the first week Adv: are heat fixing so not dealing with a live bacterial cell Dis: are heat fixing so the bacterial cell may have a bit of a distortion to it and not be the more natural shape Steps:

What is the difference between basic and acidic dyes?

basic = + acidic = -

Coccus / Cocci

circular; spherical-shaped bacterial cell

What are chromophores?

color-bearing ion

Vibrios

comma-shaped; curved rod-shaped bacterial cell

Spirillum / Spirilla

corkscrew spiral; type of bacterial cell with rigid spiral shape and external flagella

Bacillus / Bacilli

rod/cylinder; bacterial cell shape that is basically cylindrical/longer than wide

Spirochete / spirochetes

spring like/coils of spring spiral; coiled spiral-shaped bacterium that has endoflagella and flexes as it moves