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

BIO Safety

• Work with opportunistic pathogens• Must handle with extreme care

• Handle tubes by glass not cap!

Pure Culture

• Culture containing only one species• Lab 2 environmental sampling

– Did you obtain a pure culture?

http://protomag.com/assets/a-brief-history-of-staph http://www.emlab.com/s/sampling/env-report-01-2007.html

Streak Plate Method

• How it works: You physically separate bacteria• Purpose: To obtain a pure culture

– Isolated colonies• Must use aseptic technique

1 2 34

Quadrant Streak

1. Streak one loopful of organism as illustrated in#1. Try not to gouge the medium.

ApplicationWhy do we need a pure culture?

– To identify organism responsible for illness

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

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 –

Eosin Methylene Blue (EMB)

• Selective and differential media• Differentiates between 2 Gram negative organisms

– E. coli– Enterobacter aerogenes

• E. coli– Small growth with a metallic sheen

• Methylene blue selectively inhibits the growth of gram +

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?

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

Gram StainLab 5

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.

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

Figure 4.17-1 Slide flooded with crystal violet (rinsed), step 1

Slide is flooded with crystalviolet for 1 min, then rinsedwith water.

Result: All cells are stainedpurple.

Gram Stain Procedure

Figure 4.17-2 Slide flooded with iodine (rinsed), step 2

Slide is flooded with iodinefor 1 min, then rinsed with water.

Result: Iodine acts as amordant; all cells remain purple.

Figure 4.17-3 Slide flooded with solution of alcohol/acetone (rinsed), step 3

Slide is flooded with solutionof ethanol and acetone for10–30 sec, then rinsed with water.

Result: Smear is decolorized;Gram-positive cells remainpurple, but Gram-negativecells are now colorless.

Figure 4.17-4 Slide flooded with safranin (rinsed), step 4

Slide is flooded with safraninfor 1 min, then rinsed with water and blotted dry.

Result: Gram-positive cells remain purple, Gram-negativecells are pink.

• 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

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!

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