1. Safety Notes Bunsen burners> open flame
Tie long hair back
NO microscopes out until Bunsen burners are off
Safety glasses are to be worn at all times
Must be worn until everyone is done at your table

2. BIO Safety Work with opportunistic pathogens
Must handle with extreme care
Handle tubes by glass not cap!

3. Pure Culture Culture containing only one species
Lab 2 environmental sampling
Did you obtain a pure culture?
A pure culture is simply a culture containing only one bacterial species
-by looking at this image you can tell fairly easily that there is only on species on this plate based on colony morphology alone
-colony morphology are the characteristics that are used to describe bacterial colonies
some examples are round, wrinkled, complex
and they also describe the margins or side profile whether they are smooth, wavy, irregular...page 57 has more examples of the different descriptions you will learn to use
A mixed culture contains multiple species
In order to obtain a pure culture microbiologist use what is called the streak plate technique

4. Streak Plate Method How it works: You physically separate bacteria
Purpose: To obtain a pure culture
Isolated colonies
Must use aseptic technique
The streak plate method allows us to actually physically separate bacteria so they grow in isolated colonies
Why is it important to obtain a pure culture?
What if you were working in a Dr.’s office or a hospital and you were asked to perform an initial identification of a patients culture?

5. Application Why do we need a pure culture?
To identify organism responsible for illness
The streak plate technique is often one of the first steps in many microbiological assays. When your supervisor asks you to identify the organism from a suptum or urine sample as a microbiologist it will be your job to find the bad one.
And this can sometimes be difficult since If you look back on Pages of the lab book you will be reminded once again about the numerous species that are found on your body
If you don’t obtain a pure culture and you are unable to identify the bacteria the patient will not be diagnosed and treated properly.
But don’t worry in the coming weeks I will show you the tools you will need to identify unknown organisms
Other reasons why microbiologist need to work with pure cultures are so we can study the characteristics of the organisms such as:
Colony morphology such as the size and shape, metabolism what food sources it is capable of using and by products, antibiotic sensitivity determine if they are resistant or able to grow with antibiotics present, molecular techniques such as PCR to see if a certain gene is present, protein assays such as ELISA and Western Blots.
Can anyone think of any other applications for the streak plate method
I came up with:
Isolating bacterial plasmid DNA which we can talk more about later in the semester and
Isolating soil organisms

6. Specialized media Pure cultures are required for biochemical testing
Specialized media such as a selective or differential media can help in obtaining pure cultures
Selective media
Inhibits certain bacteria from growing while allowing others to grow
Differential media
Contain indicators or ingredients that allow different organisms to display a different, characteristic pattern of growth

7. MSA Selective and Differential
Selects for salt tolerance (Staphylococcus species)
Differential for Mannitol fermentation
pH indicator turns media yellow when acid by product is produced by the bacteria. The organism itself is not acidic, it “pooped” acid
S. aureus S. epidermidis –

8. Eosin Methylene Blue (EMB)
Selective and differential media
Differentiates between 2 Gram negative organisms
E. coli
Enterobacter aerogenes
Small growth with a metallic sheen
Methylene blue selectively inhibits the growth of gram +

9. Streak Plate Procedure (Demo)
Materials
Mixed culture
TSA plate
Inoculating loop
Bunsen burner
Igniter
Bacterial broth culture
We will also plate this sample on MSA and EMB
What are your expected results?
To perform the SPM you need the following materials

10. Procedure notes
Mix the cultures (only if the bacteria are settled on the bottom of tube)
LOOP> LIP> LIP> LOOP
Avoid the sizzle
Obtain ONE loopful of broth culture
Follow the quadrant streak pattern as illustrated in your lab notebook
Remember to flame the loop between streaks!
Place plate upside down
The procedure is in your lab notebook and I will give a demonstration on how to perform the technique.
First resuspend the bacterial cultures you do this by rolling between your hands. Or lightly vortex
Sterilize loop and allow to cool for a few seconds don’t sizzle
Then Flame the opening of the tube this not only helps keep the surface free of contamination but the heat creates a convection current away from the opening so airborne bacteria are less likely to settle into the tube while you have it opened contaminating your sample.
I have seen some lab techs remove the cap with the same hand and not flame the tube that is incorrect technique
Obtain a loopful of culture reflame tube opening and replace cap
Slightly lift the lid of the petri dish and spread culture in the first quad use page 55 if you can’t remember, Don’t gouge into the agar.
Flame your loop
Spread bacteria from 1st quad to 2nd
Flame loop
Etc…
Once all 4 quads are spread flame loop, tape the plate closed, label with initials, place upside down in incubation canister.
Can anyone tell me why we place the plates upside down for incubation?
Is it because bacteria don’t experience gravity?
What temperature should we incubate them?

11. Gram Stain Lab 5

12. Gram Stain
Purpose: Diagnostic test to see weather bacteria are Gram – or +
Gram + : thick peptidoglycan layer
stain purple
Gram - : thin peptidoglycan layer and an LPS layer
stain pink
Depending on the result clinicians can determine the treatment regimes, determine what battery of biochemical tests to run, and gain information about the type of infection present.
Depending on the result clinicians can determine the treatment regimes, determine what battery of biochemical tests to run, and gain information about the type of infection present.

13. Smear prep
Draw a circle on the back of slide and smear in that area
Transfer bacteria from broth to slide
Use more than one loopful for staining
Make a back up slide this means 2 stains per person
Dry on slide warmer
Heat fix
Purpose: to kill and adhere organisms to the slide for staining purposes
Bacteria side up
Don’t BBQ your bacteria

14. Figure 4.17-1 Slide flooded with crystal violet (rinsed), step 1
Gram Stain Procedure
Slide is flooded with crystal
violet for 1 min, then rinsed
with water.
Result: All cells are stained
purple.
Figure Slide flooded with crystal violet (rinsed), step 1

15. Figure 4.17-2 Slide flooded with iodine (rinsed), step 2
Slide is flooded with iodine
for 1 min, then rinsed with
water.
Result: Iodine acts as a
mordant; all cells remain
purple.

16. Slide is flooded with solution of ethanol and acetone for
Figure Slide flooded with solution of alcohol/acetone (rinsed), step 3
Slide is flooded with solution
of ethanol and acetone for
10–30 sec, then rinsed with
water.
Result: Smear is decolorized;
Gram-positive cells remain
purple, but Gram-negative
cells are now colorless.

17. Figure 4.17-4 Slide flooded with safranin (rinsed), step 4
Slide is flooded with safranin
for 1 min, then rinsed with
water and blotted dry.
Result: Gram-positive cells
remain purple, Gram-negative
cells are pink.

19. Notes: Young cells less than 24 hours are needed
Peptidoglycan begins breaking down leaving you with a mixture of pink and purple cells from a pure culture
Do not over decolorize! This is the most important step. If you over decolorize
Gram + can appear Gram – (usually you have a mixture of pink and purple cells)
Gram – would still look pink

20. Smear prep & Gram stain procedure notes
Smear preps (4 preps per person page 66)
Prepare all four of your smears using four clean slides prior to performing the Gram stains. Doing them one at a time will not allow you to finish the lab on time.
Gram Stain (4 per person page 68)
Do all at one time
Everyone must do their own!

21. Smear prep & Gram stain procedure notes
Clean slides using Bon Ami soap
Wet slide with water
Wet soap with water and rub soap on slides
Allow slide to dry with soap on
When dry rub soap off with kim wipe