Class 2Review of Class One - Microscopy andIntroduction to Aseptic Technique and MediaMrs. Sidelsky

Key Words

Parfocal and parcentric( paracentric)Parfocal means that once you have

focused on low power you should be close to focus when you increase the magnification

Parcentric – If your point of interest on a slide is centered on low power, it should remain in a central position.

Magnification

Increase the size of microorganisms by using lenses to affect the light

A maximum of 1000x can be attained by using a light microscope without sacrificing the clarity of the image

1000X means that the object is magnified by 1000 times its actual size

Managing light

Three ways to adjust lighta. Iris diaphragmb. Condenser lensc. Diopter( adjust light intensity)

Coarse and fine adjustment Use coarse adjustment only with

scanning and low Use fine adjustment only with high

and oil

Never use fine adjustment with scanning – you should not need it

Resolution

Resolution is the clarity and accuracy of the image.

When light is produced by the lamp underneath the stage it can enter the lens system through the aperture or opening from different directions

High and oil immersion have the smallest distortion of images and the highest clarity, because the light rays enter the lens system almost perpendicular to the stage.

How large are bacteria ?

Bacteria range in size from 0.1 um to 600 um( microns)

Mycoplasma are very small, but there are also nanobacteria

Epulopiscium fisheloni is the largest bacteria

Oil Immersion

In order to view prokaryote cells, it is necessary to use high magnification.

Start your focus on scanning and low. Find the best portion of the slide for study.

You want to choose a place where the cells are space so you can study the shape and arrangement

Bacterial cells – shape and arrangement

Oil Immersion (continued)

When you increase the magnification remember to adjust the lighting

You may need more light on higher magnifications

When you have focused on high power and your image is clear, turn the revolving nosepiece between high and oil

Place a drop of oil on the slide and turn your oil immersion lens gently into the drop

Remember to use the fine adjustment

Aseptic

Work to protect yourself and contain organisms under safe working conditions

Prevent contamination of cultures from external sources so that microorganisms in the Petri Dish can be identified and characterized.

Part Two

Introduction to Microbiological Techniques

Objective One-To be able to work with aseptic technique

Goals - - To be able to handle media and

cultures - To be able to transfer

microorganisms from one culture to another

Terminology

Media – Solid – Agar Trypticase Soy Agar( TSA) - Enriched

Media Nutrient Agar( NUT) Enriched Media( many) Media – brothTSA brothNUT BrothFermentation tubes- sugars

Equipment

Inoculating Loop Inoculating Needle Flaming the loop Petri Dish Streak Plate Slant Deep Broth

Media

Flaming- prevents contamination of culture

Hold Inoculating loop

Insert in flame until loop glows red

Allow to cool

Transfer of broth to broth

Steps for Transfer of Broth to Broth Hold loop or needle with dominant

hand( right ) Flame the loop Hold culture tube in left hand Remove red cap with pinkie of right hand Flame mouth of culture tube  Place loop into broth( water) Flame mouth of culture tube and close Open culture tube with broth( should be

labeled) Dip loop into new broth and mix Flame mouth of tube and close Flame loop Place to the side of your rack

Broth to slant

1. Wrap fingers of non dominant hand around the culture tube containing broth for transfer

    2. Using the pinkie finger of your dominant hand twist the red cap from the tube.  Hold in your pinkie and do not place it on the counter

3.     Pass the mouth of the culture tube across the flame

4.     Direct the inoculating needle into the broth. 5.     Flame the mouth of your broth culture tube

and replace the cap.  Place it in your rack 6.     Pick up the slant in your non dominant hand   

Part 2

Twist off the red cap 8.     Flame the mouth of the slant tube 9.     Direct the inoculating needle into the tube

and “ stab” the agar in the base( butt) 10. Withdraw on the entry line and when you

reach the surface make a simple streak along the face.

11.  Flame the mouth of the tube and replace the cap.

12. Flame your inoculating needle and replace in your rack.

Broth to streak plate  Procedure for Streaking a Plate for Isolation:

Procedure:   1.  Flame the loop and wire and streak a loopful of broth as

at A in the diagram.   2.  Reflame the loop and cool it.   3.  Streak as at B to spread the original inoculum over more of the agar.   4.  Reflame the loop and cool it.   5.  Streak as at C.   6.  Reflame the loop and cool it.   7.  Streak as at D.   8.  Label the plate and incubate it inverted.    

Go To Results of Streak Plate Lab Procedures  

Streak plate

Growth of organism over plate