Microbiology Chapter 3

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Created Sep 15, 2011
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olaswider

 

 
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1
The Five I’s:
 
•Inoculation –Addition of microorganism to supporting medium •Incubation –Proper growth...
2
How do you culture a microorganism?  
 
•To identify the agent that causes a specific disease requires that microorganisms be...
3
Step 1 to cultivate microorganism
 
–Inoculum (sample to be cultured)•Environmental specimens (water, air, soil)•Clinical...
4
Step 2 to cultivate microorganism
 
–Medium(collection of nutrients allowing growth of inoculum)•Liquid broth •Solid media...
5
Step 3 to cultivate microorganism
 
–Incubation (placing inoculated media at proper temperature for specific period of time)•Observable...
6
Clinical Specimens:
 
•A clinical specimen is a sample of human material to be examined or tested for the presence...
7
How do you properly handle a clinical specimen? (3 Steps)
 
•Must take care not to contaminate the sample with organisms from the environment or other...
8
•Obtaining Pure Cultures:
 
–Technique for isolating the suspected pathogen from normal microbiota in a culture
9
Pure culture :
 
–refers to a cultures composed of cells arising from a single progenitor (parent or original...
10
Aseptic technique:
 
refers to a set of instructions used to prevent further contamination by microbes
11
Two common pure culture isolation techniques:
 
•Streak plates •Pour plates
12
•Culture Media:
 
–Used to support the growth of microorganism or as a means of identification
13
–3 Physical States of Media
 
•Liquid •Semi solid •Solid
14
6 types of general culture media:
 
•Defined media  •Complex media  •Selective media  •Differential...
15
Physical States of Media  •Liquid:
 
–Water-based solution –Growth appears as cloudiness or turbidity
16
Physical States of Media Semisolid:
 
–Contains agar that thickens, but does not form a solid Used to determine motility
17
Physical States of Media Solid:
 
–Agar:  complex polysaccharide derived from algae –Will allow culture media to...
18
•Defined Media:
 
–Exact chemical composition is known –Used for organisms with strict need for specific...
19
•Complex media:
 
–Exact composition is not quite known –Yeast, meat, soy or protein extracts provide...
20
Selective Media:
 
•Contains substances that favor the growth of particular organisms or inhibit the...
21
Differential Media:
 
•Presence of visible changes in the media or differences in the appearance of colonies help differentiate...
22
Differential Media Blood agar-
 
differentiates between the ability of bacteria to digest, partially digest or inability to digest...
23
Differential Media Carbohydrate tubes:
 
differentiates between the ability to ferment types of sugars and the resulting products (acid or...
24
Differential Media MacConkey agar-
 
–selective for Gram – bacteria, differentiates between non-lactose and lactose fermenting Gram...
25
Anaerobic Media:
 
•Obligate anaerobes need to be cultured in the absence of oxygen –Agar Stab –Reducing media...
26
Transport Media:
 
•Special media to allow for transfer of clinical specimens –Prevent contamination...
27
Special Culture Techniques Animal and cell culture:  
 
Not all organisms will grow in media, need live cells or organism
28
Special Culture Techniques  •Enrichment culture:
 
–Selective media or technique to increase the growth of bacteria found in very small numbers...
29
•Preserving Cultures (3 steps):
 
–Refrigeration •Stores for short periods of time –Deep-freezing (-50°C to...
30
Microscopy:
 
Refers to the use of light or electrons to magnify organisms not visible with the unaided...
31
General Principles of Microscopy (4):
 
–Wavelength of radiation –Magnification –Resolution –Contrast
32
General Principles of Microscopy (4) Wavelength of Radiation:
 
•Using radiation of smaller wavelengths results in enhanced microscopy
33
General Principles of Microscopy (4) Magnification:
 
•Increase in the size of an object (number with X, read times) •Determined by multiplying...
34
General Principles of Microscopy (4) Resolution:
 
•Resolution or resolving power is the ability to distinguish between objects that are...
35
General Principles of Microscopy (4) Contrast:
 
•Refers to the differences in the intensity between two objects or between an object and...
36
Types of Microscopes (3)  Light Microscopes:
 
–Bright-field •Simple •Compound –Dark-field –Phase •Phase contrast •Differential interference...
37
Types of Microscopes (3)  Electron Microscopes:
 
–Transmission (TEM) –Scanning (SEM)
38
Types of Microscopes (3)  Probe Microscopes:
 
–Scanning Tunneling (STM) –Atomic Force (AFM)
39
Light Microscopy –Bright-field microscopes•Simple:
 
  –Contain a single magnifying lens –Similar to magnifying glass –Capable of...
40
Light Microscopy –Bright-field microscopes •Compound
 
–Series of lenses for magnification –Light passes through specimen into objective...
41
Light Microscopy –Dark-field microscopes:
 
•Best for observing pale objects •Only light rays scattered by specimen enter objective...
42
Light Microscopy  Phase microscopes:
 
•Used to examine living organisms or specimens that would be damage by attaching them to...
43
Phase microscopes (2 types)  Phase-contrast microscope:
 
»Simplest
44
Phase microscopes (2 types)  Differential interference contrast microscope (Normarski):
 
Involves prisms to split light into different colors, increasing contrast, 3Dappearance
45
Light Microscopy  Fluorescent microscopes:
 
•Direct a UV light source at specimen •Specimen emits visible light when bombarded...
46
Light Microscopy –Fluorescent microscopy •Confocal microscopes:
 
Use florescent dyes or antibobies- Uses UV lazer-Creates 3D image
47
Electron Microscopy:
 
–Electron microscopes have greater resolving power and magnification (10,000X to 100,000X)  –Detailed...
48
Two Types of Electron Microscopes  Transmission (TEM): 
 
–Specimen must be very thin –Electrons are sent through specimen, similar to that...
49
Two Types of Electron Microscopes Scanning (SEM):
 
–Specimen is coated with a metal –Electrons are sent across the surface of the specimen –Primary electrons...
50
Probe Microscopy:
 
–Magnifies more than 100,000,000 times –Detects individual molecules and atoms –Uses a...
51
2 Types Probe Microscopes Scanning tunneling microscopes:
 
•Probe is passed back and forth and slightly above specimen •Measures the flow of...
52
2 Types Probe MicroscopesAtomic force microscopes:
 
•Probe is passed along the surface of the specimen •Tip moves up and down with contour...
53
Staining, what does it do?
 
•Increases contrast and resolution by  coloring specimens with stains/dyes
54
Types of stains (4):
 
–Simple stains  –Differential stains •Gram stain •Acid-fast stain •Endospore...
55
Wet mount:
 
–Cells are suspended in liquid, one or two drops are added to slide, slide is overlaid with...
56
Hanging drop:
 
–Best for observing motility –Suspended cells are added to coverslip, coverslip...
57
How to prepare a specimen for staining:2 Steps
 
•Create a smear by spreading a thin sample of specimen on...
58
Simple Stains:
 
•Used to determine size, shape, and arrangement of cells •Composed of a single...
59
Differential Dyes:
 
•Used to distinguish between different types of cells or structures •Use more than...
60
Types of Differential Stains (3):
 
–Gram stain –Acid-fast -                 -Endospore
61
Gram Stain is used for:
 
•Used to differentiate between two large groups of microorganisms (Gram – and Gram...
62
Clinical Relevance of the Gram stain:  Presence of Gram negative organisms may indicate:
 
–Serious illnesses •Endotoxins are part of the outermost LPS layer in Gram – bacteria...
63
Gram positive bacteria:
 
–Final color: purple  –Thick layer of peptidoglycan in cell wall  –Retains...
64
Gram negative bacteria:
 
–Final color: red/ pink  –Much thinner layer of  peptidoglycan in cell wall  –Does...
65
Acid-Fast Stain:
 
-have hydrophobic layer  •Waxy layer of acid-fast bacterial cells will retain primary...
66
Endospore:
 
dehydrated structure formed inside a bacterial cell in response to harsh environmental conditions
67
Characteristics of Endospore (5):
 
–Usually found in Gram + cells  –Dormant, non-reproductive  –Tough...
68
Endospore Stain, what color does it stain?
 
•Malachite green is retained in spore •Vegetative (growing) cells will counterstain...
69
Special Stains  Negative (capsule) stain:
 
•Negative stains are repulsed by negative charges on cells •Stain the background...
70
Special Stains  Flagellar stain:
 
•Used to determine the presence, number, location and arrangement of flagella •Flagella are...
71
•Staining for Electron Microscopy
 
-stain in the form of a metal •Prevent electrons from passing through (TEM) •Excited by...


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