Unit 4 Flashcards
Culture/culturing (verb)
Process of growing bacteria in a lab
Reasons for culturing bacteria
To isolate different bacteria from a sample
To amplify number of cells
To indenting and characterize
To test for antibiotic resistance/susceptibility
For commercial reasons
Bacterial culture (noun)
Any population of bacteria grown and propagated under specific conditions in a lab/in vitro
Pure culture AKA monoculture
All bacteria in a culture are identical, therefore all originated from a single colony forming unit
Mixed culture
Contains 2 or more different bacteria (different genus/species or strain)
Indicated more than one colony forming unit present at the start
Sterile
Free of living organisms
Inoculation
The process of introducing a microorganism
Contamination
Unintentional or accidental introduction is microorganism(s)
Media
A liquid or gel designed to support growth of bacteria
Broth media
Liquid or pourable media
Solid media
Brother media that has been made into a solid with addition of gelatin or agar
Different media will be selected based on
Species cultures and reason for growing
Media types
Supportive (general purpose)
Enriched
Selective
Differential
Transport/storage
Supportive media AKA general purpose media
Supports the growth of many different organisms (not all)
Does not support growth of bacteria with strict requirements
Examples: luria broth (LB), trypticase soy agar (TSA)
Enriched media
General purpose media that has been supplemented to support bacteria that cannot grow on supportive media alone
Supplemented with different growth factors (amino acids, sugars, minerals, tissues)
Examples: blood agar, chocolate agar
Bacteria that can only grow on enriched media are termed
Fastidious (complex/complicated)
Why are selective and differential media important to clinical microbiology
Help indentifying which bacteria may be growing in a unknown sample
Can often identify genus and sometimes species
Selective media
Only grow SOME bacteria And not others, or inhibit others
Components of selective media
Antibiotics: allows only strains resistant to that antibiotic to grow
Different sugars: allow some bacteria with the metabolic enzymes to digest grow
Bile salts (MacConkey’s agar): selects for gram negative bacteria
Selenite, brilliant green dye: selects for salmonella
Differential media
Allows many organisms to grow, but highlights differences between organisms
Differences related to metabolic pathways or toxin production
Contains dyes or substrates that are only metabolized by some bacteria
Blood agar
Differential media
Used to differentiate between hemolytic and non hemolytic bacteria
Transport and storage media
Most common
Maintains and protects bacteria for long periods of time
Does not allow bacteria to grow, or grows at minimal rate
Why would we want to use transport/storage media
To prevent over growth and death phase, to have an accurate representative sample to look at later
Trypicase soy broth (TSB)
Supportive/general purpose media
For growth
Adding agar produces solid trypticase soy agar (TSA)
Blood agar
Trypicase soy agar (supportive media) with addition of 5-10% sheeps blood
Enriched and differential
Purpose:
Growth of pathogenic bacteria which are fastidious
Can determine if bacteria is hemolytic
Hemolysis
Ability to lyse (damage) RBCs
Caused by bacteria that produce hemolysin (releases nutrients from RBCs for use by the bacteria cell)
These bacteria are pathogenic
3 types of hemolysis
Alpha
Beta
Gamma
A-hemolysis (alpha)
Partial hemolysis
Hemoglobin broken down to methemoglobin (greenish zone around the colony) (bruising)
B-hemolysis (beta)
Complete hemolysis
Clear zone around the colony
B=best
Y-hemolysis (gamma)
No hemolysis
Media stays red
“Y no break down man”
Most important staining procedure in clinical microbiology
The gram stain (first step in identification)
Used to differentiate bacteria on basis of cell wall structure
Primary stain
Stain that highlights specific structure that we are looking for
Counterstain
“Non specific” stain that colors anything not bound by the primary stain
Primary stain is
Crystal violet
Enters the cell wall and distributes throughout layers of peptidoglycan
After the primary stain is
Treatment with iodine
Iodine bind to crystal violet(becomes a merger molecule that is trapped and cannot be washed away)
Structure being highlighted is the presence of multiple layers of peptidoglycan
After iodine is
Alcohol wash (decolorizer)
Alcohol shrinks peptidoglycan layers in gram positive cells (crystal violet and iodine more tightly trapped)
Alcohol damages membrane of gram negative cells (crystal violet and iodine is realized from Periplasmic space)
After alcohol wash is
Counterstain (Safranin or basic fuschin)
Stain enters the damaged outer membrane or gram negative cells and any other cells that don’t have crystal violet/iodine
Gram positive may take up some Counterstain but it is not visible against crystal viole
Cell wall size and composition of gram positive cell
Thick
20-80 layers of peptidoglycan, teichoic acid
Cell wall size and composition of gram negative cell
Thin
Outer membrane (lipopolysaccharides, phospholipids) 1-2 layers of peptidoglycan
What color are gram positive cells after staining
Purple (crystal violet)
What color are gram negative cells after staining
Pink/red (safranin)
Are gram positive cells susceptible to penicillin
Yes
Are gram negative cells susceptible to penicillin
No
Are gram positive cells resistant to physical disruption by osmotic lysis
More resistant
Are gram negative cells resistant to physical disruption by osmotic lysis
Less resistant
