Lecture 4 Flashcards
Reasons for culturing bacterai
To isolate different bacteria from a sample
To amplify the number of cells
To identify and characterize
To test for antibiotic susceptibility and resistance
For commercial reasons
Bacterial culture is
ANY population of bacteria grown and propagated under specific conditions in a laboratory
Also referred to as bacteria that are grown in vitro
Depending on the species, bacteria require different physical conditions and nutritional requirements for growth
Pure culture
AKA monoculture
ALL bacteria in that culture are identical
Therefore, all originated from single CFU
Mixed culture
Contains 2 or more different bacteria - can be different genus/species or strain
Indicates more than one CFU present at start
Sterile
free of living organisms
Inoculation
The process of introducing microorganism
Contamination
unintentional or accidental introduction of microorganisms
Culture media is and types
Media= A liquid or gel designed to support growth of bacteria
Broth media– Liquid, pourable
Solid media– Broth media that has been made into a solid with addition of gelatin or agar
Depending on the species cultured and the reason for growing, different media will be selected
Different species of bacteria have different nutritional requirements (eg. some bacteria only grow if certain types of sugar are present)
Different media support faster growth
Specific media are required to run certain tests (such as antibiotic testing)
Media types
Supportive (AKA general purpose)
Enriched
Selective
Differential
Transport /storage
Supportive media
AKA General Purpose media
Media that supports the growth of many different microorganisms
Function: To grow and propagate bacteria
Does not support bacteria with strict growth requirements
E.g., 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
Growth factors may include specific amino acids, sugars, minerals, tissues
E.g.,Blood agar, chocolate agar
Bacteria that only grow on enriched media are termed “fastidious”
Fastidious= complex/ complicated
Eg.Rickettsia,Neiserria gonorrhea, Chlamydia
Selective and differential media
Important in clinical microbiology to help identify which bacteria might be growing in an unknown sample
Can often identify the genus and occasionally can identify the spp.
Selective media
Selectively grow only some bacteria
Do not allow growth of others
E.g., promotes the growth of bacteria A while inhibiting growth of bacteria B, C and D
Examples of selective media components:
Antibiotic → selectively allows only strain resistant to that antibiotic to grow
Different types of sugars that only some bacteria have the metabolic enzymes to digest
Bile salts (MacConkey’s agar) selects for Gram negative bacteria; Gram positive bacteria cannot grow in its presence
Selenite, brilliant green dye selects for Salmonella
Differential media
Allows many organisms to grow, but highlights differences between organisms
Differences are often related to metabolic pathways or toxin production
Contain dyes or specific substrates that are only metabolized by some bacteria
Blood agar can be used to differentiate between hemolytic and non- hemolytic bacteria
Transport and storage media
Most common type of media seen in clinic
Maintains and protects bacteria for a long period of time
I.e., Between sample collection and clinical analysis
Purpose: Does not allow bacteria to grow or grows at a minimal rate
Trypticase Soy Broth (TSB)
Supportive/general purpose media
For growth
Adding agar produces a solid trypticase soy agar (TSA)
Blood agar (BA)
Trypticase soy agar (supportive media) + 5-10% whole sheep blood
Enriched , differential
Purpose:
Good for growth of pathogenic bacteria, many of which are fastidious
Can determine if bacteria is hemolytic
hemolysis
Ability to lyse (damage) RBCs
Caused by bacteria that produce hemolysin
Purpose of this toxin is to release nutrients from RBCs for use by the bacteria cell
Bacteria that cause hemolysis are more pathogenic
Can detect hemolysis by growing bacteria on blood agar and looking for distruction of the RBCs contained in the media
Alpha hemolysis
Partial hemolysis
Hemoglobin is broken down to methemoglobin → greenish zone around the colony
Beta hemolysis
Complete hemolysis
Clear zone around the colony
Gamma hemolysis
NO hemolysis
media stays red
The gram stain
Most important staining procedure in clinical microbiology
Typically, the first step in bacterial identification
Used to differentiate bacteria on the basis of cell wall structure
Primary stain
Primary stain is CRYSTAL VIOLET
Stain particles enter the cell wall and distribute throughout layers of peptidoglycan
Followed by treatment with Gram’s iodine
Iodine molecules bind to the crystal violet
Becomes a larger molecule that is trapped and cannot be washed out
Structure being highlighted is the presence of multiple layers of peptidoglycan
Alcohol wash
Cells are washed with alcohol
Alcohol shrinks the peptidoglycan layers in Gram positive cells
Crystal violet/iodine complex is more tightly trapped
Alcohol damages the outer membrane of Gram negative cells
Any crystal violet/iodine is released from the periplasmic space
