Lab Two Flashcards
How thick does the smear need to be?
Thin, but still thick enough to see some cells
what happens when the smear is too thick?
it is hard to see individual cells
What does heat fixing do?
kills the organism
adheres the organism to the slide
alters the organism so it can readily pick up stain
What is a stain?
A stain is a substance that adheres to a cell and gives it colour.
What can different stains be used for?
Different stains have different affinities for different organisms or parts of organisms and so they can be used to differentiate organisms or parts of organisms
what is the general purpose of staining?
to make bacteria contrast against the background so that they are easier to see.
What is a differential stain?
distinguish between two types of bacteria
Typically two or more reagents
what is a negative stain?
the background is stained and the living bacteria are not. Uses a single reagent
-also called indirect staining
what is a simple stain?
the dead bacterium takes up the stain and the background does not.
-also called direct staining
what is an acidic stain?
Have a negative charge and have an affinity for positive cell components. A negative dye will stay outside a negative cell wall.
what are some examples of acidic stains?
nigrosin
india ink
picric acid
what is a basic stain?
Have a positive charge and have an affinity for negative cell components. A positive dye will go inside the negatively charged cell wall
what are some examples of basic stains?
methylene blue
crystal violet
carbol fuchsin
safranin
what is a decoloriser?
Lipid solvent for gram-negative cells (alcohol dissolves the outer lipid layer, the cell looks colourless). Protein dehydrating agent is gram positive cells (cell pores shrink as they are dehydrated by the alcohol, limited or no removal of the primary stain)
what is a mordant?
often used to fix dyes in cells, making it more difficult to decolourise.
how do you heat fix a smear?
smears must be completely dry before heat fixing
The heat fix, pass the underside of a slide through a bunsen flame three times
what happens when you heat fix too little?
the smear will wash off
what happens when you heat fix too much?
the organisms may be incinerated
what are the steps to heat fix a smear from a solid media?
- place a clean slide on blotting paper
- flame loop to sterilise
- place a loopful of saline towards one end of the slide
- flame the loop again and cool
- remove a small amount of one colony and transfer to the drop of saline
- mix the cell into the saline drop and spread the suspension
- flame the loop
- allow the smear to air dry completely
- heat fix the smear
what are the steps to heat fix a smear from a liquid media?
- place a clean slide on a piece of blotting paper
- flame loop
- place a loopful of the sample towards one end of the slide
- use the loop to spread the sample
- flame the loop
- allow the smear to air dry completely
- heat fix the smear
what does a gram stain tell us?
gives information about the cell morphology and gram reaction.
what is the gram stain result for gram-positive cells?
Bacteria that retain the primary stain (crystal violet) appear purple and are gram-positive.
what is the gram stain result for gram-negative cells?
Bacteria that are decolourised and then counterstained by safranin appear pink and are designated gram-negative.
what reasons might a gram-positive (purple) cell appear gram-negative (pink)?
- because the culture is old and the cell walls have started to lose their structure
- over decolorisation with alcohol
what determines whether the cell is gram-negative or gram-positive?
The amount of peptidoglycan in the cell wall
What is the primary stain in gram staining?
crystal violet
what is the decolorising agent in gram staining?
ethanol
what is the counterstain in gram staining?
safranin
what is the mordent in gram staining?
iodine
what are the steps for gram staining?
- prepare heat-fixed sample
- place slide into crystal violet for 30 sec
- remove the slide and rinse with water
- place the slide in iodine for 30 sec
- remove and rinse with water
- drop ethanol onto the slide until no more colour is being removed
- rinse with water
- place slide into safranin for 30 seconds
- remove and rinse with water
- blot the slide dry
what is light microscopy?
refers to any microscopes that use visible light
what forms the image in a light microscope?
A series of finely ground lenses that direct light from the specimen into the objective lens
what is the image magnified by?
the ocular lens or eyepiece
how do you calculate the magnification factor?
multiply the objective lens magnification by the ocular lens magnification
What are the magnifications of low power?
4 and 10x
what is the magnification of high power?
40x
what is the magnification of oil immersion?
100x
how does the oil in an oil-immersion work?
the oil has the same refractive index as the glass of the slide and so it counteracts any refraction that may have occurred as the light moved through the lens
what does the specimen need to look like in order to form a clear image?
specimens must contrast sharply to the background to form a clear image
what are the morphological features of bacteria?
- gram reaction
- cell shape
- motility
- presence or absence of endospores
- acid fastness
what are the primary physiological tests used to identify an unknown bacterial species?
- growth conditions
- catalase activity
- oxidase activity
- glucose utilization
- the test for oxidative and fermentative metabolism
what is a domain?
all prokaryotes belong to either Bacteria or Archaea domain
what is a phylum?
a group of organisms with certain degress of morphological similarity
what is a class?
a major division in the phylum
what is an order?
a group of families whose individuals may vary in many ways
what is a family?
a collection of similar genera
in prokaryotic nomenclature, the family always ends in the suffix -aceae
what is a genus?
groups of species with common characteristics
what is a species?
the ‘basic unit’ of taxonomy, representing a specific recognised type of organism
a group of related isolates or strains
the difficulty for the taxonomist is to decide how different two isolates must be in order to be separate species of just different strains of the same species
what is a subspecies?
a division of a species; usually arises as a consequence of geographical isolation within a species
what is a strain?
a population of an organism that arises from a single organism or pure culture
what is the bacterial nomenclature?
The first name is the genus and is capitalised
the second name is the species and is NOT capitalised
what is the purpose of a presumptive identification test?
less accurate than confirmatory tests, but indicate the likely identity
what is the point of a control?
to make sure that only certain variables are affecting the outcome of the experiment. Controls ensure the result is valid.
what is a negative control?
A control that is known to give a negative result
what is a positive control?
known to give a positive result. Ensures that the conditions of the experiment are able to provide a positive result
what does a gram stain determine?
the cell morphology
what does a growth conditions test determine?
whether the bacterium is able to grow in the presence and/or absence of oxygen
what does a motility test determine?
whether the bacteria is motile or not
How do you carry out a motility test?
Prepare a wet mount of the bacteria
Observed under a microscope
what does a catalase test determine?
identifies organisms that produce the enzyme catalase
what is the function of the enzyme catalase?
it breaks down H2O2 into H2O and O2
how do you carry out the catalase test?
- hold one end of a capillary tube between the thumb and middle finger
- dip the other end of the tube into 3% hydrogen peroxide, the liquid will move up the tube via capillary action
- stopper the end of the tube you are holding with your index finger
- Touch the end holing the liquid to an isolated colony on an agar plate so as to transfer a small amount of a colony to the end of the tube
- look for vigorous bubbling in the capillary tube
how do you interpret the results of the catalase test?
bubbles- positive
no bubble- negative
What does the glucose utilization test determine?
If a species ferments glucose. If glucose can be utilized, bacterial growth will produce acid
what is the procedure for the glucose utilization test?
- heavily inoculate glucose tryptone water with the organism to be tested
- incubate at 35 degrees for 18-24 hours
how do you interpret the results from the glucose utilization test?
organisms able to ferment glucose will turn the media yellow and a gas bubble will be present in the Durham tube
what does the oxidation fermentation test determine?
indicates whether the organism ferments or oxidises carbohydrate
what is the procedure of the oxidation, and fermentation test?
- heavily inoculate two tubes of O-F media with the organism to be tested, stabbing the bottom of the tube
- to one of the tubes add 500 microlitres of paraffin oil and tighten the lid to create an anaerobic environment
- keep the lid of the other tube slightly to create an aerobic environment
- Incubate at 35 degrees for 48-72 hours
what is an API 10S test?
It is a rapid identification test
Tests for 12 different reactions using microtubes
what is the procedure for API 10S test?
- add 3ml of sterile water to the bottom of the tray to create a humid atmosphere
- Pick one single isolated colony from an 18-24 hour culture of the organism to be tested and emulsify it in 5ml of sterile water
- Mix thoroughly to prepare a homogenous suspension
- fill both tubes and cupule of the test (CIT)
- fill only the tubes (and not the cupules) of the other tests, avoid air bubbles
- overlay the tests LDC, ODC, H2S, and URE with sterile paraffin oil
- incubate at 35 degrees for 24 hours
how are the results from the API 10S test interpreted?
positive results have a numerical value (1,2 or 4)
Negative results have a value of 0
what is the 16s rRNA sequence?
A ribosomal RNA sequence is found in all microorganisms and is easy to compare
what are the advantages of S16S rRNA sequencing?
all cells contain ribosomes and ribosomal RNA
RNA genes have undergone few changes over time (are highly conserved)
Cells do not have to be cultured in the lab for sequencing
what is the process of S16S rRNA Sequencing?
- extraction of DNA from the bacterial colony
- Amplify the 16srRNA gene or regions within it (the 16s gene has 9 variable regions)
