Unit 1: Bacterial Cultivation

Question 1

Media containing known quantities of each component

Answer 1

Defined Media

Defined carbon and nitrogen sources

Can eliminate variability between experiments

Usually has narrower range of growth than complex media (not very helpful if you don’t know what you have)

Usually more expensive than complex media

Question 2

___________ media contains components not chemical defined

Answer 2

Complex media (ie yeast extract)

No exact formula

Usually has a broader range of growth than defined media

Usually less expensive than defined media

Question 3

Complex media used for growing fungi

Answer 3

Potato extract agar - bacteria won’t grow on it

Question 4

What is enriched media?

Answer 4

Complex media + growth factor(s)

Used for growing fastidious organisms (fussy or picky eaters who must have a certain factor to grow)

Question 5

What is selective media?

Answer 5

Select against unwanted organisms

Growth of unwanted organisms is inhibited by some components (ex: an antibiotic)

Only certain organisms are able to grow

Question 6

What is differential media?

Answer 6

Differentiates between organisms

Contains components that result in a visible change (color of colony, formation of precipitate etc)

Does not select for specific organisms or specifically inhibit growth

Question 7

What is MacConkey agar and how is it used?

Answer 7

Contains:
• Peptone
• *Lactose*
• Bile salts - inhibit Gram(+)
• Crystal violet - inhibit Gram(+)
• Neutral red - pH indicator

Differential AND selective

Used in enteric infections

Question 8

What are fastidious organisms?

Answer 8

Can be high maintenance, picky eaters, and are often called “fussy” - need a complex diet

Difficult to grow in vitro

Requires addition of growth factors (enriched media required)

Examples: Haemophilus and Neisseria - require hemin and NAD for growth —> will not grow on blood agar plates, only on chocolate agar (lysed red blood cells)

Question 9

Most pathogenic bacteria are ____________

Answer 9

Mesophiles - can live at ranges ~12-42˚C but optimized for 30-37˚C

Why? B/c that’s our body temp!

Lower temps: Psychrotrophs and Psychrophiles
High temps: Thermophiles and Hyperthermophiles

Question 10

Different points on the growth rate curve for environmental factors

Answer 10

MIN = minimum value of parameter that supports growth of the microbe

OPT (peak) = value of parameter that supports optimum growth of the microbe

MAX = maximum value of parameter that supports growth of the microbe (NOTE - MAX ≠ maximum growth)

Different parameters affecting growth: temp, pH, O2, nutrients)

Question 11

Low temperature challenges for bacteria

Answer 11

Loss of enzyme activity

Decrease in membrane fluidity

Question 12

High temperature challenges for bacteria

Answer 12

Denaturing of structural proteins

Denaturing of enzymes

Question 13

Diagnostic media for human pathogens usually at what pH?

Answer 13

7.0

Most pathogens affecting humans are neutrophiles - again, because they need to thrive at human body pH

Exceptions:
Acidophiles such as lactobacillus, coxiella burnetii
Alkaliphiles such as bacillus app., vibrio cholerae

Question 14

What is the meaning of obligate?

Answer 14

Without an alternative
Limited to a narrow range of a given environmental parameter

Ex: Obligate aerobes require O2

Question 15

What makes something a facultative species?

Answer 15

Has alternatives, capable of surviving in different conditions

Question 16

Classification of microbes based upon oxygen requirement

Answer 16

Obligate/strict aerobes require oxygen, have enzymes for reactive O2 species (ROS)

Microaerophile require O2 but at lower levels, have ROS

Facultative aerobe/anaerobes do not require O2 but will use it if available, have ROS

Aerotolerant anaerobes neither require nor utilize O2, but do have ROS

Obligate/strict anaerobes - O2 is toxic, do not have ROS enzymes

Question 17

Importance of ROS enzymes

Answer 17

Reactions with O2 often create reactive oxygen species (ROS) that can be toxic to cells. Without enzymes to counteract ROS, they can be lethal.

Examples of ROS enzymes: superoxide dismutase (SOD), catalase, perioxidase

Obligate anaerobes do not have ROS enzymes, so O2 is toxic

Question 18

Growth of different microbes in liquid culture, based on O2 requirements

Answer 18

Obligate aerobes - growth will be at the top of the test tube

Obligate anaerobes - growth will be at the bottom of the test tube

Facultative anaerobes - growth will be throughout the test tube, with slight clustering at the top

Question 19

Microbes that require high CO2 levels (3-5%) are classified as ….

Answer 19

Capnophiles

Examples - Neisseria, Haemophilus, Helicobacter, Capnocytophaga

Require special housing - candle jar, CO2 packet/incubator, automated systems

Question 20

Microbes that require high salt

Answer 20

Halophiles

Obligate halophiles
• Extreme (15-30%) - Archaea
• Moderate (3-15%) - pseudomonas, staph aureus - b/c on the skin (think sweat)
• Slight (0.5-3%)

Facultative/halotolerant halophiles also exist

Question 21

Microbes that require high osmolarity/sugar

Answer 21

Osmophiles

Question 22

Microbes that have developed resistance to desiccation

Answer 22

Bacillus and Clostridium via spore formation

Mycobacterium via cell wall modification (mycolic acids)

Question 23

The new gold standard for establishing the cause of an infection

Answer 23

PCR - invented 1983 by Kary Melli’s

Identifies cause of a disease using a single copy of DNA or RNA from a blood/tissue sample

Potential problems due to normal flora (microbiota)

Question 24

______________ involves a transient or permanently established microbial growth, but with NO interference with normal body functions

Answer 24

Colonization

Question 25

______________ involves a transient or permanently established microbial growth, but MAY manifest disease

Answer 25

Infection

Question 26

Abnormal condition of body structure(s) and function(s)

Answer 26

Disease Occurs when interaction leads to pathogenesis Results in damage to host Can occur without presence of microbe

Question 27

Examples of toxins from microbes causing physiological changes

Answer 27

Intoxication Clostridia and Staph species - toxins ingested from contaminated sources (food, Botox injections, drinks)

Question 28

Objective evidence of disease as noted by an observer

Answer 28

Signs Changes due to disease, observed by a clinician

Question 29

The subjective evidence of disease as sensed by the patient

Answer 29

Symptoms Changes due to disease, experienced by the patient

Question 30

_________ is sensed and observed, and therefore exists as both a sign and a symptom

Answer 30

Fever

Question 31

The five stages of disease

Answer 31

``` Incubation period Prodromal phase Invasive phase (most severe signs and symptoms) Decline phase Convalescence period ``` Severity and duration of disease and each stage varies with pathogen, virulence, and host susceptibility

Question 32

Characteristics of the Incubation (preclinical) stage

Answer 32

Pathogen has entered the body No signs or symptoms Innate immune system has not been activated Pathogen must be an infectious dose (inoculum, or number of microbes required for disease) NOT contagious!!!

Question 33

Characteristics of Prodromal (warning) stage

Answer 33

Appearance of signs and symptoms (non specific - ie dry cough and slight fever) Activation of the innate immune system Pathogen numbers increase Easily transmitted even before person realizes they are sick (Asymptomatic carriers)

Question 34

Characteristics of Acute stage

Answer 34

Aka Clinical Illness Most severe time of illness Characteristic signs/symptoms Acquired immune system has been activated Pathogen numbers are stationary Communicable diseases are easily transmitted

Question 35

Differentiating between Acute and Chronic Disease

Answer 35

Acute: signs and symptoms develop rapidly, peak and decline Chronic: Signs and symptoms persist, slow to move to decline phase

Question 36

Characteristics of the Decline stage

Answer 36

Illness still apparent but signs/symptoms dwindle Immune system activity is reduced (antibodies formed) Pathogen is cleared from host Can be contagious if individual becomes a carrier

Question 37

Characteristics of Convalescent stage

Answer 37

``` Patient returning to full health Signs/symptoms end No immune system activity to pathogen Pathogen cleared from host Not contagious (no pathogen) ```

Question 38

What are opportunistic pathogens

Answer 38

Organisms can be part of the patient’s normal flora Do not produce disease in normal setting Establish disease when introduced to unprotected site (ie blood, tissues), via injury (burns) or if immune system is compromised.

Question 39

What are virulent pathogens

Answer 39

Strict pathogens - always associated with disease | Signs/symptoms due to host inflammatory response or direct damage to or loss of tissues/organs

Question 40

How do bacterial metabolites function as mechanisms of bacterial virulence?

Answer 40

Acids, gases, or other byproducts of metabolism formed during bacterial growth serve to directly damage host tissues Ex - Streptococcus mutants produces lactic acid which results in dental caries

Question 41

How do Invasins function as mechanisms of bacterial virulence?

Answer 41

Aka “Spreading factors” Proteins or enzymes that act locally to damage host cells Affect tissue matrices and intracellular spaces (“soften”) • Allows for the spread and replication of the bacterium • Host cell/tissue damage can result in pathological characteristics • Examples: Hyaluronidase depolymerizes hyaluronic acid (connective tissue); Collagenase breaks down collagen; Neuraminidase degrades neuraminidase acid (intracellular glue); Streptokinase and Staphylokinase both act as fibrinolysins (break down blood clots)

Question 42

How do adhesins work as mechanisms of bacterial virulence

Answer 42

Bind pathogens to receptors on host cells and tissues Most bind to carbohydrate moieties (glycoproteins) Types of adhesins: • Glycocalyx material - capsule and slime layers (create biofilms) • Fimbriae and pili - attach to specific host molecules (ie - Neisseria gonorrhoeae attaches to conjunctiva) • Afimbrial adhesins - proteins associated with cell envelope

Question 43

How do toxins function as mechanisms of bacterial virulence?

Answer 43

Toxins resemble enzymes - proteins with high activity and specificity, are heat labile

Question 44

What are exotoxins?

Answer 44

Found in both gram (-) and gram (+) cells Proteins that are toxic to cells, usually direct in action Secreted into the extracellular fluid, or associated with the bacterial cell surface Bind host cell receptors

Question 45

What are enterotoxins?

Answer 45

Same as exotoxins but cause GI signs and symptoms (ie vomiting, nausea, diarrhea) Example - Rotavirus NSP4 - causes infantile diarrhea

Question 46

The 2 major categories of exo/enterotoxins

Answer 46

``` A-B Exotoxins Two domains or subunits: • A - enzymatic component, ATTACKS cell • B - BINDS toxin to host receptor After binding (B), A is transported into cell Can form toxoids (inactivated toxins) ```

Question 47

Examples of A-B exotoxins

Answer 47

Corynebacterium diptheriae stops host cell protein synthesis Clostridium botulinum prevents release of ACh at synapses —> flaccid paralysis Clostridium tetani prevents inhibitory neurotransmitter release —> spastic paralysis

Question 48

What are superantigens

Answer 48

Toxins that activate up to 40% T cells in the absence of antigen, resulting in massive release of cytokines

Question 49

Examples of membrane active exotoxins

Answer 49

Proteases - destroy protein in host cell (cell membrane) Lipases - destroy lipids in cell membrane Hemolysins - form pores in RBCs and phagocytes

Question 50

What are endotoxins?

Answer 50

LPS of gram (-) organisms Not very toxic until released O antigen - polysaccharide that is immunogenicity Lipid A component - toxicity

Question 51

Other examples of virulence mechanisms

Answer 51

1) Spread from primary site of infection - cytotoxic effects span larger area than focus of infection 2) Encapsulation - blocks phagocytosis (ex - S. pneumo) 3) Inactivation of antibody - S. pneumo secretes IgA degrading proteases, S. aureus binds to Fc region of IgG 4) Intracellular growth and escape detection - M. tuberculosis is a facultative intracellular bacterium

Question 52

What is evasion by antigenic variation?

Answer 52

Ability of microbe to change epitomes, present new antigens to immune system, or initiate new disease cycle to increase virulence

Question 53

Accumulation of genetic mutations —> alterations in protein products and variability in population

Answer 53

Antigenic Drift Forms antigenically distinct strains Example: E.coli O157:H7 - over 157 different O antigens Influenza virus - minor mutations in protein spikes, evelope

Question 54

Reassortment of viral genome in a cell infected by 2 or more strains

Answer 54

Antigenic shift Example: Influenza A - combination of two or more strains can produce a new subtype of virus —> pandemic

Question 55

Genetic conversion/shuffling due to recombination within a group of genes

Answer 55

Antigenic switching Results in new surface antigen, but no change in biological function Examples: Pili, fimbriae, surface glycoproteins Pili of N.gonnorhoeae always present but antigenic structure changes