Exam 1: Ch 2: Tools of the Lab Flashcards
Biological specimen
tissue, urine, blood, intraperitoneal fluid, semen, vaginal fluid, spinal fluid, etc
Abiotic specimen
solid, food, water, bed sheets, fomites
Inoculation
implantation of microorganisms into/onto culture medium (media)
Culture
the propagation of microorganisms w/various media
Medium/media
a nutrient used to grow microorganisms outside their natural habitat; classified according to 3 properties: physical state, chemical composition, functional types
Physical state of media
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
Liquid media
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!!!!!!!!!!
Semi-solid media
contain low % of agar (~0.4%)
o Used for: motility testing – motile bacteria can move thru it b/c it is very soft
Solid media
contain high % of agar (1-5%)
o Used for: testing biochemistry of the microbe; isolation – it enables formation of discrete colonies
Chemical composition of media
referring to whether or not the exact chemical make-up of the media is known
defined or complex
Defined media
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
Complex media
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
Functional types of media
referring to how the media will be used, or the purpose of growing the media
general purpose, differential, selective
General purpose media
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)
Differential media
chemicals added to the media for the purpose of: testing the biochemistry of the bacteria
Selective media
chemicals added to the media for the purpose of: selecting for bacteria that have a specific biochemistry
Incubate
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
Isolate
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
Colony
macroscopic cluster of cells appearing on solid medium arising from multiplication of a single cell
Streak plate
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
Loop dilution
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
Spread plate
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
Inspect
researcher or clinician observes the microbes macroscopically (holding it up in front of you to the light)
− Macroscopically – for color, size and texture
Identify
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
-SEMSelective media
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 (-))
Differential media
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
Both selective & differential media
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)
Staining
add contrast
o Both positive and negative stains can be: simple (1 stain) or differential (2+ stains)
Positive stains
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)
Negative stains
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)
Special stains
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
Smears
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
Microscopy
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)
Resolution
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
Contrast
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
Properties of light
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)
Optical microscopes
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
Electron microscopes
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
Enriched media
contains complex organic substances that some species require to grow; use this to grow fastidious bacteria
Carbohydrate fermentation media
has sugars that can be fermented and a pH indicator to show reaction; fermentation → acids → lower pH
Biochemical tests
method using other techniques to characterize cellular metabolism; can determine: nutrient requirements, products given off during growth, presence of enzymes, mechanisms or deriving energy
Oil immersion
uses oil to capture some of the light that would otherwise be lost; reduces scatter which increases resolution; used at 100x magnification
Scanning objective
40x objective; the lowest power objective
Refractive index
how contrast is measured; the degree of bending of light as it passes from one medium to another
Wet mount
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)
Heat fixation
air dried smear heated gently; kills specimen and secures it to the slide
Acidic dyes
negative charge; attract positively charged cell types; many cells repel this so they are good for negative staining
Basic dyes
positive charge; attract negatively charged cell types; many cells stain more readily w/this
Acid fast stain
differentiates acid fast bacteria (pink) from non acid fast bacteria (blue); used for medically important bacteria, fungi and protozoa
Endospore stain
dye forced by heat into resistant bodies; distinguishes btwn spores and the cells they come from
Differential stain
2 differently colored dyes (primary and counter-stain) to distinguish btwn cell types or parts
3 good things a microscope must provide
magnification, resolution, contrast
Total power of microscope
formed by the combined lenses – a combination of 2 separate lenses (objective x ocular)
Things to consider when choosing how to mount a specimen for microscopy
- Condition of specimen (living or preserved state)
- Aims of examiner (observe small structures, identify microorganisms, see movement)
- Type of microscopy available
What staining does to a specimen
imparts a control to cell/cell parts by becoming affixed on them thru a chemical reaction
Types of dyes for differential staining (Gram staining)
− Primary dye: crystal violet
− Mordant: gram’s iodine
− Decolorizer: alcohol
− Counterstain: safranin
