Lecture 4

Question 1

Reasons for culturing bacterai

Answer 1

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

Question 2

Bacterial culture is

Answer 2

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

Question 3

Pure culture

Answer 3

AKA monoculture
ALL bacteria in that culture are identical
Therefore, all originated from single CFU

Question 4

Mixed culture

Answer 4

Contains 2 or more different bacteria - can be different genus/species or strain
Indicates more than one CFU present at start

Question 5

Sterile

Answer 5

free of living organisms

Question 6

Inoculation

Answer 6

The process of introducing microorganism

Question 7

Contamination

Answer 7

unintentional or accidental introduction of microorganisms

Question 8

Culture media is and types

Answer 8

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)

Question 9

Media types

Answer 9

Supportive (AKA general purpose)
Enriched
Selective
Differential
Transport /storage

Question 10

Supportive media

Answer 10

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)

Question 11

Enriched media

Answer 11

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

Question 12

Selective and differential media

Answer 12

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.

Question 13

Selective media

Answer 13

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

Question 14

Differential media

Answer 14

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

Question 15

Transport and storage media

Answer 15

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

Question 16

Trypticase Soy Broth (TSB)

Answer 16

Supportive/general purpose media
For growth
Adding agar produces a solid trypticase soy agar (TSA)

Question 17

Blood agar (BA)

Answer 17

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

Question 18

hemolysis

Answer 18

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

Question 19

Alpha hemolysis

Answer 19

Partial hemolysis
Hemoglobin is broken down to methemoglobin → greenish zone around the colony

Question 20

Beta hemolysis

Answer 20

Complete hemolysis
Clear zone around the colony

Question 21

Gamma hemolysis

Answer 21

NO hemolysis
media stays red

Question 22

The gram stain

Answer 22

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

Question 23

Primary stain

Answer 23

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

Question 24

Alcohol wash

Answer 24

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

Question 25

Counterstain

Answer 25

Counterstain is safranin or basic fuschin
Stain enters the damaged outer membrane of Gram negative cells and any other cells that don’t have crystal violet/iodine
Gram positive cells can also take up the counterstain, but it will not be visible against the dark purple crystal violet stain