Neisseria gonorrhoeae, a diplococcus, causes gonorrhea in humans. Members of this genus produce diplococci with flattened adjacent sides.
3-74 TETRADARRANGEMENT(GRAMSTAIN, X1320)✦Micro-
coccus roseusgrows in squared packets of cells, even when they are bunched together. The normal habitat for Micrococcus species is the skin, but the ones here were obtained from culture.
Diplococcus
Streptococcus
Staphylococcus Tetrad
Sarcina
3-72 DIVISION PATTERNSAMONGCOCCI✦Diplococci have a single division plane and the cells generally occur in pairs. Strepto- cocci also have a single division plane, but the cells re main attached to form chains of variable length. If there are two perpendicular division planes, the cells form tetrads. Sarcinae have divided in three perpendicular planes to produce a regular cuboidal arrange- ment of cells. Staphylococci have divided in more than three planes to produce a characteristic grapelike cluster of cells. (Note:
Rarely will a sample be composed of just one arrangement. Repor t what you see, and emphasize the most complex arrangement.)
3-75 STREPTOCOCCUS
ARRANGEMENT(GRAMSTAIN, X1000)✦ Enterococcus faeciumis a strepto - coccus that inhabits the digestive tract of mammals. This specimen is from a broth culture (which enables the cells to form long chains) and was stained with cr ystal violet. Notice the slight elongation of these cells along the axis of the chain.
3-76 STAPHYLOCOCCUSARRANGEMENT(X1000)✦Staphylo- coccus aureusis shown in a blood smear. Note the staphylo- cocci interspersed between the er ythrocytes. S. aureusis a common oppor tunistic pathogen of humans.
3-77 STREPTOBACILLUSARRANGEMENT(CRYSTALVIOLETSTAIN, X1200)✦Bacillus megateriumis a streptobacillus. These cells were obtained from culture.
3-78 PALISADES ANDANGULARARRANGEMENTS OFARTHROBACTER(GRAMSTAIN, X1000)✦Stacking of rod-shaped cells side-by-side is the palisades arrangement (P). An angular arrangement (A) is when a pair of rods is bent where the cells join.
Cor ynebactriumalso shows these arrangements.
A
P
✦ Theory
Stains are solutions consisting of a solvent (usually water or ethanol) and a colored molecule (often a benzene derivative), the chromogen. The portion of the chromo - gen that gives it its color is the chromophore. A chromo - gen may have multiple chromophores, with each adding intensity to the color. The auxochrome is the charged portion of a chromogen and allows it to act as a dye through ionic or covalent bonds between the chromogen and the cell. Basic stains1(where the auxo chrome becomes positively charged as a result of picking up a hydrogen ion or losing a hydroxide ion) are attracted to the negative charges on the surface of most bacterial cells. Thus, the cell becomes colored (Figure 3-79). Common basic stains include methylene blue, crystal violet and safranin. Ex- amples of basic stains may be seen in Figures 3-66, 3-77, and 3-80.
Basic stains are applied to bacterial smears that have been heat-fixed. Heat-fixing kills the bacteria, makes them adhere to the slide, and coagulates cytoplasmic proteins to make them more visible. It also distorts the cells to some extent.
✦ Application
Because cytoplasm is transparent, cells usually are stained with a colored dye to make them more visible under the microscope. Then cell morphology, size, and arrangement can be determined. In a medical laboratory, these are usually de termined with a Gram stain (Exercise 3-7), but you will be using simple stains as an introduction to these.
✦ In This Exercise
Today you will learn how to prepare a bacterial smear (emulsion) and perform simple stains. Several different organisms will be supplied so you can begin to see the variety of cell morphologies and arrangements in the bacterial world. We suggest that you perform all the stains on one or two organisms (to get practice) and look at your lab partners’ stains to see the variety of cell types. Be sure that you view all the available organisms.
✦ Materials
Per Group
✦clean glass microscope slides
✦methylene blue stain
✦safranin stain
✦crystal violet stain
3-5 Simple Stains
1Notice that the term “basic” means “alkaline,” not “elementary;”
however, coincidentally, basicstains can be used for simplestaining procedures.
_ _
_ _
Cell is stained Negatively charged cell
Apply basic stain
(Positive Chromogen )++ ++ _ _ _ _ __
_ _ __
+ + + + + + ++
_ _++ _ _++ _ _++
+ + _ _++
+ + _
_ _ _ __
_ _ __
_ _ _ _ _ _ _ _ _ _ _ _
3-79 CHEMISTRY OFBASICSTAINS✦Basic stains have a positively charged chromogen (●Ⳮ), which forms an ionic bond with the negatively charged bacterial cell, thus colorizing the cell.
3-80 SAFRANINDYE IN ASIMPLESTAIN(X1000)✦This is a
simple stain using safranin, a basic stain. Notice that the stain is associated with the cells and not the background. The organ- ism is Bacillus subtilis, the type species for the genus Bacillus, grown in culture.
disposable gloves
✦squirt bottle with water
✦staining tray
✦staining screen
✦bibulous paper (or paper towels)
✦slide holder
✦compound microscope with oil objective lens and ocular micrometer
✦immersion oil
✦lens paper
✦recommended organisms:
⽧Bacillus cereus
⽧Micrococcus luteus
⽧Moraxella catarrhalis(BSL-2)
⽧Rhodospirillum rubrum
⽧Staphylococcus epidermidis
⽧Vibrio harveyi
Procedure
1 A bacterial smear (emulsion) is made prior to most staining procedures. Follow the Procedural Diagram in Figure 3-81 to prepare bacterial smears of each organism. If working in groups, each student should perform the various stains on one or two organisms, then observe each other’s slides to see the variety of cell shapes and arrangements. Prepare several emul- sions at once so they can be air drying at the same time. If staining more than one organism, you can make separate emulsions of each on the same slide.
2 Heat-fix each smear as described in Figure 3-81.
3 Following the basic staining procedure illustrated in the Procedural Diagram in Figure 3-82, prepare two slides with each stain using the following times:
crystal violet: stain for 30 to 60 seconds safranin: stain for up to 1 minute methylene blue: stain for 30 to 60 seconds Be sure to wear gloves when staining. Record your actual staining times in the table provided in the Data Sheet so you can adjust for over-staining or un- derstaining.
4 Using the oil immersion lens, observe each slide.
Record your observations of cell morphology, arrangement, and size in the chart provided on the Data Sheet.
5 Dispose of the slides and used stain according to your laboratory’s policy.
1. Place a small drop of water (not too much) on a clean slide using an inoculating loop.
If you are staining from a broth culture, begin with Step 2.
2. Aseptically add bacteria to the water.
Mix in the bacteria and spread the drop out.
Avoid spattering the emulsion as you mix.
Flame your loop when done.
3. Allow the smear to air dry.
If prepared correctly, the smear should be slightly cloudy.
4. Using a slide holder, pass the smear through the upper part of a flame two or three times.This heat-fixes the preparation. Avoid overheating the slide as aerosols may be produced.
5. Allow the slide to cool, then continue with the staining protocol.