Aseptic techniques, smear preparation and simple staining

Lab # 2Lab # 2

Bio 261, MicrobiologyBio 261, Microbiology

Prof. SantosProf. Santos

AIM

• Ex’s 9, 11, and 12

Aseptic technique

Aseptic technique refers to the reduction of contamination from the environment and other areas as the scientist is transferring bacteria from one medium to another.

Aseptic techniques

1- wipe your work area before, and after you are done working

2- If you ever spill something, please call me because it needs to be wiped immediately and the area needs to disinfected again with Lysol!

3- when dealing with microorganisms, you need to maintain sterile conditions.

Transferring cultures

1- when transferring from a liquid medium to a liquid medium, flame the mouth of the tubes and flame the loop until it is red hot. Follow the protocol on the book. I will demonstrate in class as well. The purpose is to maintain the purity and to make sure you have only a pure culture!

2- when transferring from a liquid to a solid, flame the mouth of tube, flame the loop and only open the plate slightly and gently streak the agar surface. DO NOT LEAVE THE PLATE EXPOSED TO AIR!

• Label the bottom of a plate and invert in the incubator to prevent moisture from condensing on the agar surface.

3- When transferring from a solid to liquid, flame the mouth of tube, flame the loop, and take out only a small sample of the culture.

• I will demonstrate!

Smear preparation

The goals of preparing a good smear are1- allow the cells to adhere to the surface of the

slide so that they are not washed off during staining.

2- allow the cells to adhere properly so that they do not shrink during staining otherwise distortions and artifacts can interfere with our results

3- prepare a thin smear to allow only one layer of cells to adhere otherwise you will get layers of cells on top of each other and you will not be able to examine the individual cells!

Procedure for making a bacterial smear

• From liquid mediumA- draw a target circle on the bottom of the slideB-Place 2 loopfuls of culture in the center of

target circle and spread over the entire circleC-Let it air dryD-Using a clothespin, grab slide and pass it

several times over the flame to heat kill and fix the organisms.

• From solid mediumA- Place 2 loopfuls of water in the center of target

circleB- disperse a small amount of cells with inoculating

loop in water over the entire circleC- let it air dryD- Using a clothespin, grab slide and pass it several

times over the flame to heat kill and fix the organisms.

Simple staining

• The use of a single stain to color a bacterial cells.• Common dyes used to stain cells are methylene

blue, crystal violet, and basic fuchsin.• Dyes are either negative or positive.• Since cells are negatively charged, they are best

stained with positive dyes that contain cationic chromophores or coloring bearing ions!

Procedure

• We will either stain an avirulent strain of Corynebacteruim diphtheria or E. coli.

1- prepare a good smear of the culture2- stain with methylene blue for one minute,

use staining tray!3- wash the stain off with water briefly!4- blot dry with bibulous paper5- look under the microscope

If you stained C. diphtheria, look for

1- pleomorphism, several shapes of the cells2- metachromatic granules, reddish granules of

volutin3-Palisade arrangement of the cells, picket fence

arrangement of the cells