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BI 234 > Exam 1: Ch 2: Tools of the Lab > Flashcards

Exam 1: Ch 2: Tools of the Lab Flashcards

1
Q

Biological specimen

A

tissue, urine, blood, intraperitoneal fluid, semen, vaginal fluid, spinal fluid, etc

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2
Q

Abiotic specimen

A

solid, food, water, bed sheets, fomites

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3
Q

Inoculation

A

implantation of microorganisms into/onto culture medium (media)

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4
Q

Culture

A

the propagation of microorganisms w/various media

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5
Q

Medium/media

A

a nutrient used to grow microorganisms outside their natural habitat; classified according to 3 properties: physical state, chemical composition, functional types

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6
Q

Physical state of media

A

refers to how solidified material the media is; how much agar is there?; the way we solidify is by adding agar (a complex polysaccharide)
liquid, semi solid, solid

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7
Q

Liquid media

A

TSB) – has no agar added; broths, milks or infusions
o Used for: aerobic/anaerobic, clump as they grow, etc
o Great way to gather large volumes of bacteria
o Cannot isolate bacteria using liquid media!!!!!!!!!!

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8
Q

Semi-solid media

A

contain low % of agar (~0.4%)

o Used for: motility testing – motile bacteria can move thru it b/c it is very soft

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9
Q

Solid media

A

contain high % of agar (1-5%)

o Used for: testing biochemistry of the microbe; isolation – it enables formation of discrete colonies

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10
Q

Chemical composition of media

A

referring to whether or not the exact chemical make-up of the media is known
defined or complex

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11
Q

Defined media

A

synthetic media; media whose compositions are precisely chemically defined
o Contain pure and inorganic compound – nothing collected from nature in this media (when collected from nature you cant tell the exact makeup)
o Molecular content specified by an exact formula

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12
Q

Complex media

A

contains ingredients that aren’t chemically defined or pure (many contain animal, plant or yeast extracts)
o We don’t know exactly what’s in it b/c they contain natural components
o Ex of additives: ground tissue or cells, blood, yeast digests, milk

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13
Q

Functional types of media

A

referring to how the media will be used, or the purpose of growing the media
general purpose, differential, selective

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14
Q

General purpose media

A

used to grow a broad spectrum of bacterial species
o Used as: first step in growing up samples from patients and formites
o Purpose: to allow every bacteria/microbe on that plate to grow up – pipe dream: the only way we have the ability to know microbes exist is to be able to grow them up (20 microbes on swab, only 18 grow up = didn’t even know those other 2 existed)

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15
Q

Differential media

A

chemicals added to the media for the purpose of: testing the biochemistry of the bacteria

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16
Q

Selective media

A

chemicals added to the media for the purpose of: selecting for bacteria that have a specific biochemistry

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17
Q

Incubate

A

incubator: chamber where temp is controlled
− Usual lab propagation temps fall btwn 20-40 degrees C
− Atmospheric gases such as O2 and CO2 may be required for the growth of certain microbes
− During incubation, microbes grow and multiply – producing visible growth in the media

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18
Q

Isolate

A

based on concept that if an individual cell is separated from other cells on a nutrient surface, it will form a colony
3 basic methods for isolating (all of which require: medium w/firm surface, a petri dish, inoculating tools): streak plate, loop dilution, spread plate

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19
Q

Colony

A

macroscopic cluster of cells appearing on solid medium arising from multiplication of a single cell

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20
Q

Streak plate

A

streaking overlapping lines on media; areas of overlap is where you’re diluting it out – if you don’t dilute it all out, you wont be able to grab a single colony
o Method only works if the spreading tool is resterilized after each of steps 1-4

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21
Q

Loop dilution

A

diluting loop into liquid media and placing sample on the solid petri dish
o Method only works if spreading tool is resterilized after each of steps 1-3

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22
Q

Spread plate

A

use different dilutions of the microbial sample; take liquid w/dropper and put on plate then use spreading tool to form a “lawn” (spreading out all around and should go to edges)
o Don’t sterilize in btwn

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23
Q

Inspect

A

researcher or clinician observes the microbes macroscopically (holding it up in front of you to the light)
− Macroscopically – for color, size and texture

24
Q

Identify

A 
determine identity of unknown microbe using various tests (biochemical, immunological, DNA analysis)
Selective media
-pH
-antibiotic
-gram + and -
Differential media
-blood agar
  -alpha-hemolysis
  -beta-hemolysis
  -gamma-hemolysis
-carb utilization tubes
Both
-MacConkey agar
-MSA
Staining
-positive
  -simple positive
  -differential positive
      -gram stain
-negative
-special
  -capsule 
  -flagellar
  -endospore
-smears
Microscopy
-resolution
-contrast
-properties of light
-optical microscopes
  -bright field
  -dark field 
  -phase contrast
  -fluorescent
  -confocal
-electron microscopes
  -TEM
  -SEM
25
Q

Selective media

A 
enables (selects) one type of bacteria to grow by the addition of a chemical that inhibits the growth of particular biochemical traits of a bacteria; select for a bacteria that has a certain attribute
o	pH – 7.0 is ideal for bacteria but fungus like acidic environments so you test for those
o	Antibiotic – selecting for bacteria that carry a resistant gene
o	Gram (+) or Gram (-) – media that only allows for growth of one or the other type of bacteria (spread E.coli and S.aureus on gram (-) and only E.coli shows up = E.coli is gram (-))
26
Q

Differential media

A

shows different reactions (colony color, media change, etc) to determine the biochemistry of the bacteria
o Blood agar – tests whether the bacteria breaks down erythrocytes
• Alpha-hemolysis – a partiallysis and will look greenish due to reaction w/hemoglobin
• Beta-hemolysis – complete breakdown of the erythrocyte
• Gamma-hemolysis – indicates an inability for that bacteria to break down erythrocytes
o Carbohydrate utilization tubes – used to see is the bacteria in question use a particular carb as fuel for metabolism
• Can also get info on: gas producing this carb, ability to metabolize this carb in +O2 and -O2 environments

27
Q

Both selective & differential media

A

give you even more info; can also be used to isolate particular bacteria from a mixed sample
o MacConkey Agar – selects for gram (-) and indicates if the bacterium ferments lactose
• Tells us that:
• Since E. coli grows and has pink/red colonies, it is gram (-) and is able to ferment lactose
• S. aureus is gram (+)
• S. enterica grows and has colorless colonies = gram (-) and not able to utilize lactose
o Mannitol salt agar (MSA) – selects for gram (+) bacteria by addition of 7.5% salt
• Differentiates btwn bacteria that are mannitol fermenters and not w/the addition of mannitol and a pH indicator
• When mannitol is fermented, the pH lowers and the media turns yellow
• The only ones that can grow w/lots of salt are those w/thick cell walls (gram + has thick peptidoglycan layer, gram – has thin wall)

28
Q

Staining

A

add contrast

o Both positive and negative stains can be: simple (1 stain) or differential (2+ stains)

29
Q

Positive stains

A

dye binds to specimen
• Simple positive stains – typically use these which bind to the specimen due to negatively charged DNA (methylene blue & crystal violet)
• Differential positive stains – allow us to differentiate btwn different typs of bacteria
• Gram stain – positive vs negative; most frequently used stain (what we did in lab: CV → iodine → alcohol → safranin)

30
Q

Negative stains

A

dye doesn’t bind to specimen, but rather around it (wont be attracted to cell but will be attracted to the environment the cell is in)

31
Q

Special stains

A

used to identify special structures that some bacteria possess
• Capsule stain – capsule doesn’t stain so we stain the background; negative stain; capsules allow bacteria to hide from immune system
• Flagellar stain – very difficult stain
• Endospore stain – difficult to stain b/c is it made of thick hard to penetrate spore wall; spores can hang out much longer in harsh environments

32
Q

Smears

A

staining requires many steps where liquids are applied to slides and washed off; for this reason bacteria must be affixed to the slide
1. Smear bacteria onto slide from broth or agar – too thick = cant see thru specimen, too thin = cant find any bacteria (thin is better than thick)
2. Air dry – don’t want too large of a smear or it will take too long to air dry; leaving any liquid on will burn the bacteria when you apply heat
3. Heat fix – pass slide thru flame quickly 3-4 times; heat fix too little = organisms may wash off slide, too much = organisms may be destroyed
• Heat fixation: kill organisms, adheres specimen to slide, promotes stainability of specimen

33
Q

Microscopy

A

after stained they must be visualized by microscope; w/out microscope it would be impossible to characterize the morphology of bacteria
o Measure based on metric system (we’ll be looking at bacteria ranging from 1um-10um)

34
Q

Resolution

A

capacity to separate 2 adjacent objects from one another; the human eye can resolve 2 objects that are no closer than 0.2mm apart → the light microscope increases this to 0.2um
• Increased resolution = more likely to see what’s actually there
• Increase resolution by decreasing wavelength – the smaller the wavelength is compared to the object you are looking at, the better the image
• Only way to increase resolution is to change light

35
Q

Contrast

A

dark vs light; the difference btwn the intensity of light and dark in the microscopic image
• Effected by: staining the sample & the amt of light that is allowed thru the sample

36
Q

Properties of light

A

light has the ability to:
• A) reflect – some light is reflected off the slide
• B) transmit – what you are seeing when you look thru the microscope
• C) absorb – the darker portions of the sample are absorbing the light
• D) refract – as you magnify the specimen more and more, this will decrease resolution (why we use oil emersion – this decreases the refraction that occurs when the light hits the working space between the objective and the slide)

37
Q

Optical microscopes

A

uses electromagnetic spectrum; all have max magnification of 1000x-2000x
• Bright-field – most commonly used in labs and classes; observe live or preserved stained specimens
• Dark-field – observe live unstained specimens; view and outline of specimens
• Phase-contrast – observe live specimens; view internal cellular detail
• Fluorescent – UV radiation causes emission of visible light from a fluorescent dye
• Confocal – combines fluorescence or unstained images to form a 3D image

38
Q

Electron microscopes

A

don’t use light waves, but rather a fine electron beam to scan the sample; magnification of 100,000x-650,000x; increases resolution up to 0.5nm
• Transmission electron microscopy (TEM) – can view internal structures of cells
• False-color scanning electron microscope (SEM) – can create 3D images

39
Q

Enriched media

A

contains complex organic substances that some species require to grow; use this to grow fastidious bacteria

40
Q

Carbohydrate fermentation media

A

has sugars that can be fermented and a pH indicator to show reaction; fermentation → acids → lower pH

41
Q

Biochemical tests

A

method using other techniques to characterize cellular metabolism; can determine: nutrient requirements, products given off during growth, presence of enzymes, mechanisms or deriving energy

42
Q

Oil immersion

A

uses oil to capture some of the light that would otherwise be lost; reduces scatter which increases resolution; used at 100x magnification

43
Q

Scanning objective

A

40x objective; the lowest power objective

44
Q

Refractive index

A

how contrast is measured; the degree of bending of light as it passes from one medium to another

45
Q

Wet mount

A

1-2 drops of a culture places on a slide and overlaid w/cover slip; provides good assessment of physical characteristics (size, color, shape, motility)

46
Q

Heat fixation

A

air dried smear heated gently; kills specimen and secures it to the slide

47
Q

Acidic dyes

A

negative charge; attract positively charged cell types; many cells repel this so they are good for negative staining

48
Q

Basic dyes

A

positive charge; attract negatively charged cell types; many cells stain more readily w/this

49
Q

Acid fast stain

A

differentiates acid fast bacteria (pink) from non acid fast bacteria (blue); used for medically important bacteria, fungi and protozoa

50
Q

Endospore stain

A

dye forced by heat into resistant bodies; distinguishes btwn spores and the cells they come from

51
Q

Differential stain

A

2 differently colored dyes (primary and counter-stain) to distinguish btwn cell types or parts

52
Q

3 good things a microscope must provide

A

magnification, resolution, contrast

53
Q

Total power of microscope

A

formed by the combined lenses – a combination of 2 separate lenses (objective x ocular)

54
Q

Things to consider when choosing how to mount a specimen for microscopy

A
  1. Condition of specimen (living or preserved state)
  2. Aims of examiner (observe small structures, identify microorganisms, see movement)
  3. Type of microscopy available
55
Q

What staining does to a specimen

A

imparts a control to cell/cell parts by becoming affixed on them thru a chemical reaction

56
Q

Types of dyes for differential staining (Gram staining)

A

− Primary dye: crystal violet
− Mordant: gram’s iodine
− Decolorizer: alcohol
− Counterstain: safranin

BI 234 (16 decks)

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