Lab 2 Flashcards
Colony
Visible growth created by multiple rounds of bacterial reproduction (cloning)
*each colony represents a single cell
Aseptic?
Aseptic technique?
Disinfect?
Sterile?
Contaminate?
Aseptic -free of living pathogens
Aseptic technique - set of procedures to prevent contamination
Disinfect - to reduce microbial numbers to safe levels
Sterile - complete removal of all microbes, including spores
Contaminate - to introduce unwanted microbes
What aseptic techniques do we follow in our lab?
- Disinefect work area
- Wash hands after cleaning the bench
- Properly sterilize inoculating loops and needle
*Only bring culture out when need it
*don’t work over top of items
*Dispose of things properly
Pure culture?
Mixed culture?
Contaminated?
Pure culture - Culture containing only 1 bacteria
Mixed culture - Contains more than 1 type of organism
Contaminated - unwanted organisms as well as desired organism
Streak plate technique
common pure culturing technique
thins out small inoculation of bacterial cells over the surface of the entire plate
*separate individual cells from one another that will form colonies, which will consist of clones
Staining
Process of adding contrast to microbes
Positive stain
Simple stain
Differential stain
Using positively charged chromogens (basic) that adhere to bacterial cells (negative charge)
Simple stain - use a single chromogen to color cells the same color
Differential stain - uses 2 or more chromogens based on their biochemical composition
Smear prep
Label slide
Plate culture - 1 drop of saline on the slide, transfer a small amount of plate growth to the drop and swirl it
Broth culture - mix the culture and transfer 1-2 loopfuls to the slide
Allow to air dry
Gently heat - hold glass slide in front of incinerator 5-6 times
let cool
Gram stain
Gram-positive
Gram-negative
A differential stain that uses various dyes
Gram-positive - a bacterium with a thick peptidoglycan layer and no outer layer
Gram-negative - a bacterium with a thin peptidoglycan layer and outer membrane
Gram stain process
- Prepare a smear of bacteria
- Apply chrystal violet, 30 sec and wash off
- Apply grams iodine. Mordant. sets stain. 1 min
- Decolorize with grams alcohol 2-5 sec
- Counterstain with Safranin. 30sec to 1 min. wash
- Blot to remove excess water and air dry
beta-hemolysis (β-hemolysis)
alpha-hemolysis (α-hemolysis)
gamma-hemolysis (γ-hemolysis)
beta-hemolysis (β-hemolysis) - beta -complete lysis, the clear area around colony growth
alpha-hemolysis (α-hemolysis) - alpha - incomplete lysis of red blood cells, green area around colony growth
gamma-hemolysis (γ-hemolysis) - gamma - no red blood cell lysis
The 3 basic shapes of bacteria
Coccus -
Bacillus -
Vibrio-
Coccobacillus -
Spirillum -
Coccus - round or spherical
- *Bacillus** - rods
- *Vibrio**- curved rods
- *Coccobacillus** - short rods
Spirillum - spirals, corkscrew
Common mistakes for gram stains
- smear prep too thick
- Over/under decolorizations
- Culture age, if over 16-18hours
Mordant
Forms an insoluble chemical complex
a substance, typically an inorganic oxide, that combines with a dye or stain and thereby fixes it in a material.
What is the purpose of flamming the culture tubes prior to entry with loops?
to produce convection currents to prevent microbes from the air from entering the culture
What will disinfection of the bench kill?
not kill?
Will kill most vegetative cells and viruses but will not destroy endospores
What is degerming?
Sanitization?
Disinfecting?
Sterilizing?
Degerming - hand washing
Sanitization - to reduce or eliminate pathogenic agents (such as bacteria) on the surfaces of (something)
Disinfecting - reduce microbes
Sterilizing - complete removal of microbes
How does an autoclave work?
Medical tools and equipment are placed inside an autoclave. The lid is sealed, air is removed from the autoclave, and then steam is pumped into the vessel. Heat and pressure are maintained long enough to kill microorganisms and bacteria in order to sterilize medical tools.
What is one major difference between serratia marcescens and Escherichia coli?
E coli - either motile or non motile, no red pigment
Serratia marcescens - motile and red pigment
What color is gram positive?
What color is gram negative?
Gram positive is purple
Gram negative is pink
Describe the gram stain reagents and their purpose?
Crystal violet - stains both positive and negative peptidoglycan layers
Grams iodine - mordant that forms an insoluable chemical complex with crystal violet
Grams alcohol - removed chrytsal violet iodine complex from gram negative making them colorless. Shirnks pores of gram positive trapping the dye. Decolorization
Safranin - stains decolorized gram negative pink to red.
why is the gram stain a differential stain?
used to differentiate between gram positive organisms and gram negative organisms.
Why is the decolorization step the most important?
it can remove color and give false positive or negative
why is the gram stain the most important in first step of IDing a unknown bacteria?
allows for the determination of the Gram reaction, morphology, and arrangement of the organism.
