Microbiology

Microbiology

Science in Motion

Summer Biology Teacher

Workshop 2004

Aseptic Technique and Staining

Techniques in Microbiology

 Background Information:

 Aseptic Technique

 Negative Stain

 Smear Preparation

 Simple Stain

 Gram Stain

Aseptic Technique



Demonstration

 Will demonstrate smear preparations

 Bacteria are smeared on a clean slide and allowed to

air dry

 Slide is then passed through a flame which kills the

bacteria and fixes them to the slide

 If done properly, bacteria will remain on slide throughout

staining processes

Negative Stain

 Use Nigrosin Stain to stain SLIDE NOT

organisms

 Background should appear dark gray and

organisms should appear opaque or

translucent against the background

 Used for morphological studies

Simple Stain

 Bacteria must be stained in order to be

observed

 Many different dyes are available to stain:

 Carbol Fushion (Raspberry Red) 10 sec

 Crystal Violet (Violet) 1 min

 Methylene Blue (Blue) 1 min

 Safranin (Pink) 1 min

 Malachite Green (Green) 1 min

Gram Stain

 Used to identify unknown species of bacteria

 Gram reaction is based on the structure of

the bacterial cell wall

 Gram + Crystal violet stain is trapped under

peptidoglycan layer

 Gram – The outer membrane prevents crystal

violet stain from reaching peptioglycan layer. The

outer membrane is then permeabilized by alcohol

(acetone) treatment, and the pink safranin stain is

trapped in the peptidoglycan layer.

Bacterial Cell Wall Diagrams

Gram Stain Procedure

Analysis of Hand Washing

Techniques

 Hand washing is the beginning of infection

control.

 Use Aseptic Technique to wash hands at ALL

TIMES to reduce infections

 Each group has a different soap sample so

that we may compare results

 Use the same soap for both parts of the lab

Inhibition of Bacteria –

Antibiotics and Antiseptics

 Alexander Fleming first discovered penicillin (by

accident) and thus lead to the understanding of

bacterial inhibition

 Antibiotics inhibit or destroy bacterial cells in

different ways

 Some inhibit the cell from producing peptidoglycan to

protect cell walls. Results in a weak cell wall and cell will

eventually rupture.

 Others inhibit the production of proteins at the ribosomes

within the cell.

 Still others affect the way bacterial ribosomes read mRNA

which leads to errors in protein structure.

Antibiotics and Antiseptics for

Experiment

 Antibiotics  Antiseptics

 (E) Erythromycin  (Bet) Betadyne

 (T) Tetracycline  (Bio) Biotene

 (S) Streptomycin  (Via) Viadent

 (N) Novobiocin  (Bac) Bactine

 (C) Chloramphenicol  (Lis) Listerine

Effect of UV Light on Microbial

Growth

 The effect of UV light negatively affects most

bacteria

 Occurs most readily between 260 and 270nm.

 The absorption of UV light causes the

production of thymine-thymine dimers

 Nucleotides fail to form bonds with dimers, thus

causing DNA replication to stop.

 Most damage is caused by the cell trying to repair itself

 Heavily damaged DNA attempt SOS repair

 Eventually results in the production of new DNA strands with

a greater number of misplaced bases

 This mutation leads to faulty protein synthesis and

eventually death

Hand Washing Lab Tips

 Make sure to label plates with group number

 Expose Plate A and Plate B at the same time

in appropriate light boxes. All groups will

expose under the same UV light

 After Plate A is finished remove plate and

expose Plate C.

Microbes in the Environment

 This lab demonstrates approximate numbers

of microorganisms growing in different

locations in the environment

 It does not attempt to identify particular species

 Each group is assigned 3 locations and 1 of

their choosing. Be Creative!!!

 Make sure to follow proper aseptic technique

 Document sample source in Data Table 1

The “Sizzler” – Bacterial

Population Counts

 Use SPC (standard plate count) to determine

the number of bacteria in a culture sample

 Procedure

 Diluting organisms from your source using sterile

water blanks

 Incubation of plates

 Analyze a plate with 30-300 colonies

 Conduct a calculation to determine the number of

organisms per milliliter of sample

The “Sizzler” – Helpful Hints

 Tiffany will dispense out pipettes. When you

need a new pipette see her.

 Read the directions CAREFULLY there are a

number of dilutions. If you mess up the first

one, you’ve messed up ALL of them.

 Demonstrate proper shaking technique

 Elbow remains on lab table