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Biology homework help>Microbiology Lab Report- Gram Staining, Simple Staining, Negative Staining

 

Introduction to ScienceExperiment 2Negative Staining Experiment Inventory

Materials 2 “Negative slides” from Experiment 1

Nigrosin

Candle

Matches

*Sink or Disposable Plastic Container

*10% Bleach Solution

*Isopropyl Alcohol

Labware 1 Clean Microscope Slide

Reusable Metal Inoculation Loop

Parafilm®

1 Pair of Disposable Gloves

*Lab Notebook (optional)

*Scissors

*Timer

Note: You must provide the materials listed in *red.

EXPERIMENT 2: NEGATIVE STAINING

Negative staining provides a more detailed assessment of a microbe’s morphology and helps prevent any distortion of the mi- robe’s ultrastructure by the staining procedure. It also makes the outline of the cells highly visible. Nigrosin and bacterial cells are negatively charged. Therefore, negative staining works because like charges repel each other, preventing the cells from taking up the stain.

PROCEDURE

Note: If you are keeping a lab notebook, record the date, time, and experiment title on a fresh page before you begin.

1. Over a sink or disposable plastic container, place 1 – 2 drops of nigrosin near the end of one of the “Negative” slides.

2. Sterilize your inoculation loop with your candle.

A. Pour 70% isopropyl alcohol into a 100 mL beaker to a depth of 2 – 4 cm. Place the cap back on the bottle, and position it

far out of the way.

B. Light your candle and set it aside. Be very cautious that your candle is safely positioned away from the beaker of isopro-

pl alcohol.

C. Dip the inoculation loop into the isopropyl alcohol for 10 seconds. Once you’ve put the inoculation loop in the alcohol,

keep it angled down so that no alcohol drips back onto your hand.

D. Without touching the inoculation loop to anything, carefully pass the end of the inoculation loop through the flame several

times.

E. Extinguish the flame when complete.

3. Use your sterile inoculating loop to transfer a colony from the culture plate that corresponds with the slide you just prepared,

and gently mix the microbes with the nigrosin.

4. Place the clean slide at about a 45° angle next to the nigrosin, and use the slide to gently spread the dye over the specimen

(Figure 10).

Figure 10: Use a clean slide set at a 45˚ angle to spread the nigrosin across the slide.

5. Repeat Steps 1 and 2 with the second “Negative” slide.

6. Air-dry the dye-stained samples for approximately 10 minutes.

7. If you have a microscope available, observe the stained slide

under increasing the magnification, and record what you see at

each magnification in Table 2 focusing on morphology and ar-

arrangement of the cells utilizing terms learned in this lab. If there

is no microscope is available, refer to Figure 11 and take a photo-

graph of your slide if required by your instructor. If keeping a lab

notebook, print out Table 2, and tape it into your lab notebook, or

re-create it by hand.

Figure 11: Isolated bacteria negatively stained with nigrosin. Image credit Rieg et al., 2010 (https://wwwnc.cdc.gov/eid/article/16/3/09-1457_article)

8. Place your slide in a disposable plastic container, and pour

bleach over the surface until the sample is completely covered/

saturated. Allow the sample to soak in the bleach for approxi-

mately 20 minutes, and then rinse the bleach down the sink with running water.

9. Wrap the bacterial slide in Parafilm®, and dispose of it in the trash.

Data SheetExperiment 2 Data Sheet Table 2: Experiment 2 Staining Observations

Stain Used:

Observations

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