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Microbial Pathogenesis > Lecture 2 Sterilization Culture Serology (E1) > Flashcards

Lecture 2 Sterilization Culture Serology (E1) Flashcards

Ch 3, 4, 5, 6, 16

1
Q

The five I’s

A

Inoculation- Start from sample
Isolation- Colony on solid media, one kind of microbe, PURE CULTURE
Inspection- Expansion and growth of microbes under proper conditions
Inspection- Observation of characteristics (data)
Identification: Use of data, correlation, ID organism, diagnosis of disease

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2
Q

Sterilization

A

any process that eliminates, removes, kills, or deactivates all forms of life and other biological agents

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3
Q

Diagnostics

A
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4
Q

Antibiotics

A
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5
Q

Sample Handling

A

Procurement- Get enough and put it in the right container document the source
Stabilization- Media, fixatives, drying, atmosphere etc
Storage- Can cold chain be maintained? Does it have to be? What is the shelf life? How should the sample be stored?
Shipment- Does your carrier meet the needs of the sample? Cold chain, tracking, chain of custody?
Use- Was the test preformed properly?
Data- FDA, HIPPA, FERPA etc

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6
Q

Streak Plate Method

A

-What iv done before
Each streak dilutes the sample
Sterilize loop between streaks
End up with colonies from a single bacteria (Axenic- Pure Culture)

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7
Q

The Pour Plate Method

A

First get pure colony
Grow in liquid culture
Make serial dilutions
How many/ ml in the original sample?
6.7x10^4 - 8x10^4

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8
Q

Solid Media

A

agar or gelatin base plus nutrient components, useful for isolation pure colonies. Can be combined with selective or differential agents

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9
Q

Liquid Media

A

Broths of various types, growth detection, differentiate oxygen requirements

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10
Q

Semi-solid Media

A

specialty media, measure motility

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11
Q

Simple Media

A

all chemical components exactly defined. Usually meant to grow limited repertoire of microbes

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12
Q

Complex Media

A

Uses extracts and proteolytic digests of animal tissues. Meant to grow a wide range of microbes

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13
Q

Key ingredients in Media

A

carbon, nitrogen, sulfur, phosphorous, metal ions, etc

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14
Q

General (complex) Media

A

grows many types of bacteria

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15
Q

Selective Media

A

Selects for and/or against growth of specific bacteria with the use of specific inhibitors. Used to recover/identify specific types of organisms from mixed culture
Accomplished in several ways:
Unique carbon source (very few other bacteria can use it)
Dyes
Antibiotics
Salts
Other inhibitors that broadly affect many (but not all) bacteria

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16
Q

Differential Media

A

Cleverly designed media that will “indicate” the use of specific metabolic pathways (i.e. use of specific carbon sources, presence of certain enzymes, antibiotics, etc)
Differentiate closely related organisms
Certain dyes or chemicals in the media yield characteristic color changes of growth patterns
Used in diagnostic, medical, environmental, food, and dairy laboratories
Media commonly combine functions and be both differential and selective

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17
Q

Blood agar (nonselective)

A

Recovery of bacteria and fungi

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18
Q

Chocolate agar (nonselective)

A

Recovery of bacteria including Haemophilus and Neisseria gonorrhoeae

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19
Q

Thioglycolate broth (nonselective)

A

Enrichment broth for anaerobic bacteria

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20
Q

MacConkey agar (selective, differential)

A

Selective for gram-negative bacteria; differential for lactose fermenting species

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21
Q

Mannitol salt agar (selective, differential)

A

Selective fro staphylococci; differential for Staphylococcus aureus

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22
Q

Xylose lysine deoxycholate agar (selective, differential)

A

Selective, differential agar for Salmonella and Shigella in enteric cultures

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23
Q

CHROMagar (selective, differential)

A

Selective, differential for selected bacteria and yeasts

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24
Q

Buffered charcoal yeast extract (BYCE) agar (specialized)

A

Recovery of Legionella and Nocardia

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25
Q

Cystine-tellurite agar (specialized)

A

Recovery of Corynbacterium diphtheriae

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26
Q

Triple Sugar Iron Media

A

Contains 3 sugars, iron, phenol red
Starts to get yellow and black ish as acid production slowly increases, lastly acid production with H2S

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27
Q

Columbia CNA

A

with 5% Sheep blood agar
Purpose: Medium for Gram+ selection and differentiation
Antibiotics colistin, nalidixic acid (CNA) select against Gram-
Sheep blood differentiates according to hemolytic reaction (glows light around gram + with hemolysis) no growth with Gram -
Hemolysis:
Gamma- none
Alpha- some
Beta- lots

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28
Q

Mannitol Salts Agar

A

Purpose: Medium for Staphylococcus selection and differentiation 7.5% NaCl selects against most common bacteria, Staph can survive pH indicators differentiate Staph spp.(grows pink) based on ability to ferment Mannitol
Staph aureus (grows yellow)

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29
Q

Eosin Methylene Blue Agar

A

Purpose: Medium for Gram(-) selection and differentiation
Sugars (lactose, sucrose) select for enteric bacteria
Dyes select against Gram(+) bacteria and differentiate fermenters
Gram(-) Strong Lactose fermenter (blueish green)
Gram(-) Lactose Fermenter (purpleish)
Gram(-) Lactose non-fermenter (light pink on edges)

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30
Q

Xylose Lysine Desoxycholate Agar Purpose

A

Purpose: Medium for Gram(-) selection and differentiation, used to isolate Shigella and Providencia
Species in fecal samples based on their ability to/ to-not ferment xylose, lactose or sucrose or reduce sulfur
Sugars differentiate fermenters(low pH)
Lysine will select for decarboxylase positive bacteria (high pH)
Thiosulfate and iron differentiate sulfur reducers
Desoxycholate selects against Gram (+) bacteria
Timing is important

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31
Q

Citrate Agar

A

Purpose: Select for bacteria able to use Citrate as sole carbon source
Differentiate Enterobacteriaceae from other Gram (-) rods
Needs two things: cell entry, utilization
Ammonium phosphate- sole nitrogen source (needed to make all amino acids, DNA, RNA bases)
Blue, growth (+)
No blue, some growth
Control tube, no growth

32
Q

MacConkey’s Agar

A

Purpose: selective and differential
Selectively isolate Gram(-) and enteric bacilli
Differentiate them based on lactose fermentation
-Crystal violet and bile salts inhibit Gram(+)
Allows for the selection and isolation of Gram(-)
Lactose fermenters detected by acid end products (pH indicator neutral red)
Varients- sorbitol, NaCl, etc
Lactose Fermenter low pH (pink)
Lactose non-fermenter high pH (yellow)

33
Q

Deep Stab Culture

A

Complex, general media
Differentiate with oxygen gradient
No growth on top- Obligate anaerobe
Growth top to bottom- Facultative anaerobe
Growth only at top- Obligate aerobe

34
Q

Aerotolerance

A

ability or inability to live in the presence of oxygen

35
Q

Obligate aerobes

A

Require oxygen for respiration

36
Q

Facultative anaerobes

A

Can grow with or without oxygen

37
Q

Aerotolerant anaerobes

A

Do not utilize O2, tolerate atmospheric levels

38
Q

Microareophiles

A

Tolerate and need low levels of oxygen

39
Q

Obligate anaerobes

A

Cannot tolerate even low levels of oxygen

40
Q

Capnophiles

A

Require elevated carbon dioxide levels

41
Q

Gram Stain (+) or (-)

A

Most commonly used stain in microbiology (e.g. Gram-positive, Gram-Negative) Double stain, decolorizing step. Based on cell wall chemistry

42
Q

Wright-Giemsa

A

For blood parasites, viral and chlamydial inclusion bodies, and Borrelia
Toxoplasma, Pneumocystis and Rickettsia spp. Polychromatic stain (methylene blue, azure B and eosin Y).
-Giemsa stain combines methylene blue and eosin. Eosin ions(-) stain basic components- orange to pink, other dyes(+) stain acidic cell structures-Blue to purple.
Protozoan trophozoites have a red nucleus and grayish-blue cytoplasm; intracellular yeasts and inclusion bodies typically stain blue; rickettsiae, chlamydiae and Pneumocystis spp. stain purple.

43
Q

Ziehl-Neelsen Stain (Acid-Fast stain)

A

Mycobacteria and other acid-fast organisms. Organisms stain with basic carbol-fuchsin- resist decolorization with acid-alkali solutions.
Counterstained with methylene blue. Organisms appear red against light blue background. Uptake of carbol-fuchsin requires heating specimen
-Hot acid-fast stain-

44
Q

Kinyoun Stain (Acid-Fast Stain)

A

Some principle as Ziehl-Neelsen stain
-Cold Acid-Fast stain- no heating

45
Q

Modified acid-fast stain

A

Weak decolorizing agent is used with any of the three acid-fast stains listed. Mycobacteria are strongly acid-fast, other organisms stain weaker (e.g. Nocardia, Rhodococcus, Tsukamurella. Gordonia, Cyrptosporidium, Isospora, Sarcocystis and Cyclospora) are referred to as partially acid-fast

46
Q

10% KOH (acid-fast stain)

A

KOH dissolves proteinaceous material, facilitate detection of fungal elements. Lactophenol cotton blue added- increase contrast

47
Q

India ink (acid-fast stain)

A

Constrast dye. Cryptococcus spp. in cerebrospinal fluid etc. Polysaccharide capsule of Cryptococcus spp. excludes ink, creating halo around yeast cell.

48
Q

Calcofluor white stain (acid-fast stain)

A

Fungal elements and Pneumocystis spp. Stain binds to cellulose and chitin in cell walls; mix dye with KOH (replace KOH stain)

49
Q

Direct fluorescent antibody stain (acid-fast stain)

A

Antibodies complexed with fluorescent molecules. Antibody binds to specific epitopes, detection by fluorescence. Useful for detecting or identifying many organisms (Streptococcus pyogenes, Bordetella, Francisella, Chlyamdia, Influenza virus, herpes simplex virus) Sensitivity and specificity– number of organisms and quality of antibodies used in reagents

50
Q

Antiseptic

A

Typically a chemical agent that is applied to living tissue to kill microbes. It is not intended to harm the host, only the microbe. Therefore, antiseptics are less toxic than disinfectants when used on inanimate objects

51
Q

Antisepsis

A

A process involving the destruction or inhibition of micro-organisms in living tissue thereby limiting or preventing the harmful effects of infection. Does not mean ALL the microorgranisms are destroyed

52
Q

Autoclave

A

A device for the application of Moist heat (steam) above the normal-atmosphere boiling point of water

53
Q

Decontamination

A

The killing of organisms or removal of contamination after use, with no quantitative implication, generally referring to procedures for making items “safe” before disposal

54
Q

Disinfectant

A

A germicide that inactivates virtually all recognized pathogenic microogranisms but not necessarily all microbial forms. They may not be effective against bacterial spores. Not generally safe for use on living tissues

55
Q

….static

A

agent needs to be present to maintain action removal of agent allows microbe reproduction

56
Q

…cidal

A

Agent needs adequate contact time for permanent action agent can be removed and sterility maintained

57
Q

Filtration

A

liquids, gases
Can sterilize without heat or other harsh treatment. Pore size of 0.2um normally used. Viruses are more difficult to filter

58
Q

Chemicals

A

Multiple types of solvents. Denature proteins (requires water), disrupt membranes, oxidizing agents. No perfect chemical-microbes are differentially susceptiple

59
Q

Moist Heat (autoclaving)

A

Method of choice for sterilization in most labs
Can kill all microbes, spores and viruses (timing)
Mechanism- hydrolysis and denaturing of cellular proteins
Pressurized steam has a high latent heat

60
Q

Dry Heat (Flaming, baking)

A

No steam (water), protein hydrolysis cant take place
Kills by oxidation of cellular components
Requires more energy, higher temperatures are required

61
Q

Contact Time

A

Moist heat has much shorter kill time (Heat of vaporization)
When using a disinfectant contact time is very important

62
Q

Evaporation time

A

The problem with some disinfectants is that they evaporate before the disinfectant time

63
Q

ELISA/EIA

A

Antibody Detection
At each step:
Block non-desired binding with excess non-interfering protein/detergent
Allow binding to achieve equilibrium
Wash away unbound excess reagents between steps
Purpose: Quantitation of antigen or antibody
clinical examples: Viral antigen(rotavirus); viral antibody (anti-HIV)

64
Q

Antigen Capture

A
  1. Capture antibody
    +specimen
  2. Second antiviral antibody
    Detector antibody usually different than capture antibody
  3. Antiimmunoglobulin enzyme
  4. Anti-detector antibody, enzyme
    +substrate
  5. to color change
65
Q

PCR- three basic steps

A

Denaturation:
Heat to 95C to separate in to two strands
Cool to between 50C and 65C
Priming:
Added primers bind to the complementary strand of test DNA
Prepares the two strands for synthesis
Extension:
72C
Thermostable DNA polymerase adds nucleotides to primers and extends the strand DNA
DNA amplification, can then be analyzed and used

66
Q

IFA

A
67
Q

Southern Blot

A

Sample:
–DNA Restriction digest
–PCR product
Load Gel (electrophoresis)
Separate DNA by size
Transfer to membrane
Detect with specific probes
DNA detection

68
Q

Restriction Enzyme “Chemical Knives”

A

Bacterial enzymes that cut BOTH strands of DNA at a specific sequence
Tools and techniques for basic elements and applications of genetic engineering
Heat-denatured DNA- Strands melt apart
Slowly cool- Strands reform
Annealing- Re-connection of strands
Dependent upon salt concentration and percent identity (sequence match)
Whole genome difficult to work with solution :Restriction enzymes

69
Q

Immunofluorescence

A

Purpose: detection and localization of antigen
Clinical examples: Viral antigen in biopsy (e.g rabies, herpers simplex virus)

70
Q

Immunohistochemistry

A

In situ localization of CMV using genetic probe
Distinguish cells that have “it” or “not”
Biotin-labeled-CMV-specific-DNA probe
Develop with streptavidin/enzyme conjugate+substrate
Localize things in situ

71
Q

Flow cytometry

A

Stain cells with fluorescent Ab (internal or external)
Count
Separate
Sort (Collect separate groups)

72
Q

Western Blot

A

Purpose: Detection of antigen- specific antibody or antigen
Clinical examples: Confirmation of anti-HIV seropositivity (antibody)
Separate antigens via electric field (SDS page)
Blot onto nitrocellulose paper (capillary or electro-transfer)
Detect antigen with antibody
Develop with precipitating or luminescent substrate
Visualize bands-compare to standards
-Takes longer, used to confirm ELISA assays(HIV, LYME)

73
Q

MALDI-TOF assay

A

Uses a laser energy absorbing matrix to create ions from large molecules
Spot protein in matrix, vaporize solvent
Laser ionizes proteins
Spectrophotometer can identify signals
Uses:
Rapid identification of proteins, bacteria, etc. from a complex mix
Determine the molar mass distribution
Identification of micro-organisms such as bacteria or fungi (usually pure colony)
Quick, diagnostic tool that does not require sequencing (individual proteins in a sea of other proteins

74
Q

Genetic engineering

A

Six applications and topics
Tools and techniques
-Restriction enzymes
Methods in recombinant DNA technology
-Polymerase chain reaction
-sequencing
Biochemical products of recombinant DNA technology
-Immune treatments, hormones, vaccines, antibodies
Genetically modified organisms
-Food (Vitamin A in rice), Herbicide resistance, Insect resistance
Genetic treatments
-Personalized Cancer therapy, Retinal diseases
Genome analysis
-Complete sequences of organisms

75
Q

Reverse Transcriptase

A

Enzyme that is used when converting RNA into DNA (from retrovirus- necessary for retrovirus life cycle RNA to DNA)
-mRNA can be converted back to DNA
-mRNA represents what is happening in a cell (which genes are on or off)
Copied DNA is referred to as complementary DNA or cDNA
Oct 1975 David Baltimore, Renato Dulbecco and Howard Temin are awarded Nobel prize, how tumor viruses interact with genetic material

76
Q

RFLP Restriction Fragment Length Polymorphisms

A

Differences in the cutting pattern of large DNA sequences by specific restriction endonuclease will give rise to RFLP’s
Comparison of RFLP between organisms (or people) shows genetic differences

77
Q

Restriction Enzymes=Restriction Endonucleases

A

Enzymes that cut DNA at specific sites
Can recognize and clip at palindromes (sequence)
Cleaves DNA creating a restriction fragment with “sticky ends”
Fragments can be joined with other DNA having similar “sticky ends”

Microbial Pathogenesis (11 decks)

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