Download Dr.-Santos-Microbiology-lab-notes-2016

Microbiology lab review
Lab Safety
Please read over the safety rules found on the preface.
You should know the words primary and secondary containment.
 Primary containment is the protection of personnel and the immediate
surrounding area from exposure to infectious agents.
 Secondary containment is the protection of the environment external to the lab.
This is provided by a combination of facility design and operational practices.
 You should know the 4 levels of Bio-safety!
 On roman numeral xii, you should be familiar with the four BSL levels we
classified organisms we use in the lab.
 BSL-1 not likely to pose a disease risk to healthy adults, know some examples
 BSL-2 poses a moderate risk to healthy adults; unlikely to spread throughout the
community; effectively treatment readily available
 BSL-3 can spread disease in healthy adults; may spread to community, effective
treatment readily available
 BSL-4 can cause disease in healthy adults; poses a lethal risk and does not
respond to vaccines or anti-microbial agents.
Lab #1 Exercises 1, 2 and 3
Please go over
 The compound light microscope and the parts of the microscope.
Part of the microscope
A- Body tube- connects the eyepiece with the rotating nosepiece.
B-eye piece- the lens the observer looks through.
C-rotating nosepiece- holds the objectives and allows you to switch from one to the other.
D-objective lens- the lens found on the nosepiece that magnifies the image. Some
microscopes are binocular. You can change the distance between the two lenses by
simple pulling apart or pushing together the oculars.
Most microscopes contain three; the low power, high power, and the oil immersion lens.
*To determine the total magnification power, multiply the power of the eyepiece with the
power of the objective lens.
For example; if the eyepiece is 10x and you are using the high power, which is 40x, then
the total magnifying power is 400x. So, it means you are looking at the object 400x
closer.
E-coarse adjustment knob-allows you to move the stage closer to the objective lens and
focus the specimen. When you first place a slide in the microscope, you should always
start with low power and focus with the coarse adjustment.
F-fine adjustment knob-allows you to move the stage very slowly and finely focus the
specimen under high power. Keep in mind that when you move to high power; the
amount of light passing through will diminish. You might need to adjust the diaphragm.
G-stage- broad flat platform where you place the slide.
H-Mechanical stage- a clamping device used to hold and move slide around on the stage.
I-diaphragm- a circular flat wheel underneath the stage that allows you to control the
amount of light passing through.
J-arm- used to carry the microscope and for support.
K-light source- either a mirror or a light bulb.
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Resolution- the ability of a microscope to separate and show two points that are
very close together. A good microscope has high resolution and magnification. If
you buy one of those cheap toy microscopes, it might allow you to see an object
400x closer but it has weak resolution. You will see everything blurry.
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The three lens system of the microscope ocular, objective and condenser
Know why we need oil when we use the 100x objective
Know resolution, numerical aperture, and the limit of resolution in most light
microscope (.2um)
Know steps taken to maximize resolving power such as use a blue filter; keep
condenser at highest level, the diaphragm should not be stopped down too much,
use oil immersion oil when using the 100x lens to minimize the bending of light
rays since oil has the same refractive index as glass.
When we first use a microscope, start with the low power dry objective, focus
with the coarse adjustment and center your specimen.
Never use oil for the dry objectives.
The best microscope to use too view unstained cells is the phase contrast
microscope.
When viewing the letter “e” slide, the image appears backward and upside down
due to a series of mirrors that adjust what is viewed.
The three color threads slide was used to illustrate depth of field.
The electron microscope uses a beam of electrons to create an enlarged image of
the specimen.
On binocular microscopes, one must be able to change the distance between the
oculars and to make diopter changes for eye differences. On most microscopes,
the interocular distance is changed by simply pulling apart or pushing together the
oculars.
To make diopter adjustments, one focuses first with the right eye only. Without
touching the focusing knobs, diopter adjustments are then made on the left eye by
turning the diopter adjustment ring on the left ocular until a sharp image is seen.
One should now be able to see sharp images with both eyes.
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Lab #2 exercises 9, 10 and 11
Please go over
 3 reasons for using aseptic techniques.
 Know the difference between primary and secondary containment.
 The general procedure for aseptic technique including work area disinfection,
loops and needles, culture tube flaming and inoculation, final flaming of loop
or needle, Petri dish inoculation, and final work area disinfection.
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When inoculating a Petri dish, raise the cover and hold it diagonally over the
plate to protect the surface from any contamination from the environment.
Go over the steps needed to transfer broth culture to another broth culture,
from broth to agar and agar to agar.
Know the three reasons for preparing a good smear.
Know the steps in preparing a smear.
Know simple staining and why we stain cells (they are clear or colorless)
Know the difference between basic and negative staining
Know what a chromophore is (colored bearing ion)
Know examples of basic and negative dyes
Basic dyes are used to stain the cells since cells tend to be negatively charged
and negative dyes tend to be used to stain the outside since they repel.
In exercise 11, we stained Corynebacterium diphtheriae using a basic stain
(methylene blue). You were told to look for pleomorphism, metachromatic
granules, and palisade arrangement of cells.
Lab # 3 exercises 14 and 16
Please go over
 This lab was very important because you guys learned how to do the Gram
stain. The Gram stain is a differential stain used to distinguish between
gram positive and gram negative bacteria. This is base on the biochemistry
of the cell wall.
 Gram – bacteria have a thin layer of peptidoglycan while gram + bacterial
have a thicker layer of peptidoglycan.
 Please know the steps and importance of each step.
Step 1 make a smear of the sample and heat fix
Step 2 the primary stain, crystal violet for 20 seconds. Gram – and gram +
cells will pick up this stain and appear purple.
Step 3 Apply the mordant or Gram’s iodine for 1 minute. This forms a tight
complex with the crystal violet in gram + cells. Both cells still appear purple.
Step 4 the decolorizer is used to remove the crystal violet/Iodine fro the gram
– cells. The decolorizer ethyl alcohol is applied for 20 seconds. The gram +
cells continue to appear purple while the others have become colorless.
Step 5 the counter stain safranin is applied for 1 minute. This is used to stain
the gram – cells. At this point they will appear pink.
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When doing the Gram stain, it is important to use fresh cultures to minimize
false results such as a gram + staining pink due to the fact that it’s so old it has
problems picking up the crystal violet. Also keep in mind that gram – never
convert to gram +.
It is critical to prepare a thin smear to allow you to see single cells instead of
layers of cells superimposed on top of each other.
Know the reason we use acid fast staining. The reason is that members of the
genus Mycobacterium and some members of the genus Nordaria have a layer
of mycolic acid that prevents them from being properly stained.
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The important thing is that the primary stain used is Carbolfuchsin is applied
over heat. This allows the stain to penetrate the layer of mycolic acid.
The counter stain used is methylene blue.
The acid fast cells tend to appear red and the non acid fast cells appear blue.
Lab # 4 exercise 14 and 16
Please go over
 The important thing about exercise 14 is that the capsule prevents ordinary
staining from taking place. The capsule found in bacteria such as Streptococcus
mutans and Streptococcus pneumoniae help them colonize and avoid destruction
by the Immune system.
 The capsule is composed primarily of polysaccharides but some capsules have
polypeptides with unique amino acids.
 Heat fixing cells after a smear will destroy the capsule and not fixing at all is no
good because the cells will jut right off.
 But using a combined method of simple stain and negative staining, we can stain
the capsule properly.
 Know the steps involved especially the India ink used as the negative stain and
the simple stain used (crystal violet).
 We looked at slides of Klebsiella pneumoniae for this exercise.
 For the spore stain, we use either the Schaeffer- Fulton method or the Dorner
method.
 Spores are resistant structures or a resting stage of certain species of bacteria
belonging to the Bacillus and Clostridium genera. Spores can only be killed by
autoclaving at 121 degrees Celsius.
 Since spores are highly resistant, it is hard for a stain to penetrate them. Heat must
be applied during staining.
 In the Schaeffer- Fulton method, the spores are stained with malachite green and
the vegetative cell is stained with safranin.
 In the Dorner method, the spore is red and the vegetative cell is colorless. The
stain used is Carbolfuchsin and nigrosin is used to create a dark background for
contrast.
Lab # 5 exercises 19, 10, 7, and 8
Please go over
 Know the difference between complex and defined media. Complex media
contains a variety of compounds needed by the organism to grow but the exact
composition is not known. In a defined medium, we know exactly what is in it.
They are useful in cultivating very fastidious organisms with strict requirements.
 Know the different between autotroph and heterotroph.
 Chemoorganotrophs- derive energy from the breakdown of organic molecules by
respiration or fermentation.
 Chemolithotrophs- oxidize inorganic ions such as nitrate or Iron to obtain energy.
Examples are nitrifying and Iron bacteria.
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Photoautotrophs- contain pigments such as chlorophyll to capture energy from the
sun and convert it to chemical energy stored in sugars. No energy source is
supplied in medium since the energy is supplied in the form of light. Examples
are purple sulfur bacteria and cyanobacteria.
Photoheterotrophs- these organisms derive energy from the sun but their carbon
source is derived form organic molecules such a glutamate or succinate. Example
is the purple nonsulfur bacteria.
Please know the nutritional requirements of bacteria. These are
carbon source
nitrogen source
vitamins
water
energy source
growth factors
Please know that a selective medium allows one type of organism to grow and
inhibit others. Example EMB medium. This allows gram – bacteria to grow and
inhibits gram +.
A differential medium allows a certain type of bacterium to take on an appearance
that distinguishes it from others. For example, S. aureus when grown on Mannitol
salt agar will produce yellow colonies since they ferment the sugar mannitol to
cause a change in pH. The phenol indicator changes from red to yellow due to the
acid formation.
Know that media can be liquid (broth) or solid (agar) or some like SIM medium
can be semi solid to detect motility!
In exercise 9 please remember the importance of a pure culture and a colony. To
obtain a pure culture we pick a single colony only!!!
2 methods are used to obtain a pure culture, streak and pour plate method.
In exercise 9, you were given a mixed culture of S. marcescens, E. coli, and M.
luteus. Which one gives you red colonies? White colonies? Small yellow
colonies?
Know the importance of the quadrant method for streaking. Why do you only dip
once into the culture tube?
The important thing about exercise 6 is to show you that bacteria are everywhere.
If you remember or if you were here, you were given different media to expose to
the environment in different ways. You were also given a sterile cotton swab
applicator to rub any surface like your cell phone and then place in the medium.
The following week you observed turbidity. This indicated growth.
Know the three basic shapes of bacteria, rod, sphere and spiral.
As far as exercise 7 is concerned, just know basic properties of fungi such as
1. are eukaryote
2. non-photosynthetic
3. lack tissue differentiation
4. have cell walls of chitin
5. propagate by spores
6. unicellular fungi are yeast
7. Molds have microscopic intertwining filaments called hyphae. A mass of
hyphae forma a mycelium. A septa is a cell wall that separates the hyphae
into individual compartments.
8. Sabouraud agar is used to grow Fungi. It contains peptones and a low pH
of 5.6 to inhibit bacterial growth.
Lab # 6 exercises 21 and 26
Please go over
 Please know the five groups of bacteria based on air requirement.
1. obligate aerobe
2. obligate anaerobe
3. facultative anaerobe
4. microaerophiles
5. aerotolerant organisms
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Know figure 19.1 indicating where the different organisms would grow.
Know the different types of media that were used in this experiment such a
TGYA or shake tubes, FTM, and Brewer’s anaerobic agar.
 Remember that the TGYA tubes were inoculated in the liquid state and then
allowed to solidify. Remember that the water bath was set at 45 degrees Celsius.
Why?
 Know the components of the FTM tubes such as glucose, cystine, and sodium
thioglycollate. Very important, there is a dye called resazurin that turns pink in
the presence of oxygen. The tube will appear pink on top since oxygen level is
higher on top. There is also some agar that will help localize the anaerobes on the
bottom. Why?
 Know the setup for the gaspak anaerobic jar. The important things to remember
are
1. The gaspak envelope provided hydrogen gas that reacts with the oxygen in
the jar to form water and carbon dioxide. This removes the oxygen
providing an anaerobic condition.
2. An indicator strip was placed to tell us when there is no oxygen. The
methylene blue strip should become colorless.
3. The lids should be tightly sealed.
As far as exercise 26 is concerned, know E.coli is by far the most common cause of
urinary tract infections.
A urine sample is examined for color, odor, turbidity, pH, mucus, blood or pus. All
these might indicate infection.
Know what we did in 26.2 where you transferred .01 ml of a urine sample and then
streaked it on a plate. The following week, you counted the colonies and then by
multiplying by 100 one can determine the original count.
Also remember that you left one sample at room temp and one was refrigerated.
Why?
Lab # 7 exercises 27, 36, and 37
Please go over
 The Kirby Bauer method to determine antibiotic effect.
 Know the difference between an antibiotic and antimicrobial agent
 Know zone of inhibition and how to measure it.
 The recommended medium is Mueller Hinton II agar.
 Disks containing the appropriate antibiotics are placed on the agar and the
following week the zone of inhibition is measured. The greater the diameter, the
more effective the antibiotic is or the more sensitive the bacterium is to that
particular antibiotic.
 In exercise #37, you used different antiseptic agents and tested their effectiveness.
Examples are phenol, Lysol, Iodine, and formaldehyde. You soaked half of a
paper disk into the antiseptic and placed it on the agar and the following week you
measured the radius to determine effectiveness.
 Know what a sanitizer is.
 Know the difference between bacteriocidal and bacteriostatic.
 Know what a sterilant is.
 Antibiotics vary in their effectiveness against various pathogenic bacteria.
 Why are certain gram – bacteria more resistant than gram + bacteria to antibiotics
that attack cytoplasmic targets?
 As far as exercise 27 is concerned know the 4 types of Neisseria species and what
they cause.
Organism
pathogenicity
N. gonorrhoeae
STD Gonorrhea
N. meningiditis
meningitis
N. sicca
Normal flora of respiratory tract
N. flavescens
Normal flora of respiratory tract
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Neisseria gonorrhoeae and Neisseria meningitidis
They are very sensitive to environmental conditions outside the body.
Both N. gonorrhoeae and N. meningitidis are easily destroyed in specimens or
samples that are;
1-Delayed in transit to the laboratory
0
2-Kept in temperature too far below or above 35 C
3-Heavily contaminated with normal flora
4-Not provided with an adequate supply of carbon dioxide
The Spirochetes (27.2)
Basic properties:
1- Slender, coiled organism
2- Axial filament allows motion
3- Thin cell wall
4- Hard to stain
3 genera you need to know!
1- Treponema- syphilis (STD)
2- Borrelia- lyme disease (skin)
3- Leptospira- leptospirosis ( urinary system, kidney failure)
(bacon lettuce tomato)!
Lab # 8 Midterm examination
Lab #9 exercises 41
Please go over
 For this lab, it is important to understand the difference between oxidation and
fermentation. Basically what you were trying to determine is whether your
organism carries out respiratory metabolism or fermentation.
 Know the difference between catabolism and anabolism in terms of metabolism.
 Know what exoenzymes are.
 The following fermentation tests were done
1. O/F glucose
2. specific sugars such as glucose, lactose and mannitol
3. mixed acid fermentation
4. butanediol fermentation
5. citrate fermentation test
O/F glucose (green colored liquid medium)
1. You were given E. coli (facultative), P. aeroginosa (oxidative), and unknown to
inoculate into this medium.
2. Each organism was inoculated into 2 O/F glucose tubes. One of them was layered
with mineral oil to maintain anaerobic condition.
3. Interpretation chart. Yellow would indicate a positive reaction
organism
With mineral oil
No mineral oil
interpretation
P. aeroginosa
Green
yellow
oxidative
E. coli
Yellow
Yellow
facultative
unknown
Yellow
yellow
facultative
You should understand why the color changes took place!
Specific sugars such a glucose, mannitol, and lactose.
1. These were red in color due to phenol red indicator and they also contained a
durham tube to see if gas was produced.
2. A color change to yellow indicated a positive result. Red would mean sugar was
not fermented.
3. If liquid got displaced in durham tube it indicates gas formation
Mixed acid fermentation
1. Some organism can ferment sugars and produce acids such as lactic, acetic,
succinic and formic acid.
2. To test for this use MR-VP medium and then add a couple of drops of methyl red
indicator. A red color means a positive result.
3- E.coli, Proteus, Salmonella and Aeromonas tend to be positive for mixed acid
fermentation.
2, 3 Butanediol fermentation
1. Some organisms ferment sugars and do not form acidic products but form neutral
end products such as 2, 3 butanediol.
2. We grow the organism in MR-VP medium and then add Barritt's reagent A (alpha
naphthol) and B (KOH). We incubate at room temp for 30 minutes. The 2, 3
butanediol is oxidized to acetoin giving us a pink color.
3. Control organism used was Enterobacter
Citrate fermentation test
1. Some bacteria can use citrate as their sole carbon source. Some bacteria can
cleave citrate into oxaloacetate and pyruvate. These intermediates are then
fermented.
2. The medium used is Simmons Citrate agar. It is a green colored medium that
contains citrate and ammonium salts to serve a nitrogen source. The ammonium
salts are broken down and ammonia is produced as a waste product. Ammonia is
basic and raises the pH of the medium causing a color change to blue.
3. A blue color change indicates positive. Green is negative.
4. Control organism used was Enterobacter
The following oxidative tests were done.
1. oxidase
2. catalase
Nitrate reduction was also analyzed
Oxidase
1. oxidative organisms have the enzyme cytochrome oxidase that allows for the
formation of metabolic water by allowing the transfer of electrons from reduced
cytochrome C to oxygen.
2. We used an artificial electron acceptor called N, N, N’, N’- Tetramethyl- pphylenediamine which changes from yellow to purple when electrons are
transferred from reduced cytochrome c to the artificial acceptor.
3- we are looking for a color change to purple to indicate our organism is oxidative.
4- control organism used was Pseudomonas
Catalase test
1. This enzyme is found in oxidative organisms that allow it to break down
hydrogen peroxide into water and oxygen.
2. We detect the enzyme by adding a couple of drops of peroxide into a glass
slide with our specimen.
3. We look for gas bubbles to indicate the breakdown of hydrogen peroxide.
4. Control organism used was S. aureus
Nitrate reduction
1. Some facultative organisms can undergo nitrate respiration in which nitrate serves
as a terminal electron acceptor.
2. We use reagent A and reagent B to detect reduction.
3. reagent A is sulfanilic acid
4. reagent b is dimethyl –alpha- Napthylamine
5. We look for a red color to indicate a positive result.
The important thing about exercise #41 is that organisms as a result of metabolism
leave behind a chemical trail. We can use indictors to detect these end products and
we can identify them based on this.
6- Control organism used was E. coli
Lab #10 exercises 42 and 43
Please go over
 The important thing in this lab is to understand that we can identify an
organism or bacterium based on its physiological or biochemical properties.
 In exercise 42, we looked for a particular enzyme. If the organism had the
enzyme it was able to metabolize the substrate in the medium creating a color
change. We look for the byproducts of the reaction. Meaning it leaves behind
a chemical “fingerprint”. This is important. It allows us to identify organisms
in the case of infection.
Enzyme
Medium used
Color change
By product detected
Amylase
Starch agar plate
Add iodine look for We look for clear
(B. subtilis)
clear zone due to
zone
starch degradation
Proteases
Skim milk plate
Look for clear zone Clear zone of
(B.subtilis)
due to degradation
degradation
of casein protein
Lipases
Spirit blue agar
We look for a dark
Blue precipitation or
(S.aureus)
blue precipitation
sometimes just
due to lowering of
depletion of fat
pH as a result of
droplets on the agar.
fatty acids being
Tryptophanase
(E.coli)
Tryptone broth
Urease
(P.vulgaris)
Urea agar slant
Phenylalanine
deaminase
Phenylalanine agar
(P.vulgaris)
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released
We add kovac’s
reagent to detect the
indole ring
produced.
Tryptophan is
broken down into
indole, pyruvate and
ammonia. We detect
the indole.
When urea is broken We detect the raise
down it releases
in pH due to the
ammonia and
ammonia being
carbon dioxide. The released.
ammonia raises the
pH and the phenol
red indicator causes
a color change from
yellow to bright
pink
We look for a
Phenylalanine is
greenish precipitate broken down by the
after the addition of enzyme into
10% ferric chloride ammonia and
phenylpyruvic acid.
The ferric chloride
reacts with the
phenylpyruvic acid
to yield a greenish
color.
Exercise 43 was important because it allowed us to use media that can
multitask! Meaning that more than one physiological property can be
detected. Think about it, it saves us time and reagents and money!
Three multiple test media were used! They were Kliger’s Iron medium, SIM,
and Litmus milk.
The Kliger’s medium contains 1%lactose, 0.1%glucose and the amino acid
cysteine, peptones, ferrous salts and the pH indicator phenol red.
The important thing to remember about it is that it is a slant that must be
stabbed. Very easy, if your organism ferments both glucose and lactose, both
the slant and butt will be yellow.
If the organism ferments only glucose, then the slant is red and the butt is
yellow.
If everything is red, then your organism does not ferment either one. It just
means you need to do more test with other sugars!
If you see a black precipitation, this means hydrogen sulfide was produced!
Procedure
1- inoculate one Iron kliger’s agar with P. vulgaris and another with E. coli and
another with your unknown
SIM
 This medium is cool because it allows us to detect three things
1) motility since medium is semi solid (0.7% agar). When you stab it, if your
organism is motile it should diffuse all over. If your organism is non motile like
shigella, it should just grow on the stab. The salmonella some of you guys inoculated
last week is motile and should diffuse. 2) SIM allows us to detect hydrogen sulfide
production by looking for a black precipitate. This is due to the breakdown of
cysteine.
3) The breakdown of tryptophan in the medium allows for the formation of an indole
ring. We can detect using drops of Kovac’s reagent.
Procedure
1- Obtain 4 SIM tubes. Inoculate with E.coli, S. aureus, P. vulgaris and your
unknown.
2- E.coli is + for indole, S. aureus is – for motility, and P. vulgaris is + for
Hydrogen sulfide production.
Litmus milk
 Litmus milk contains 10% powdered skim milk, litmus pH indicator and the pH
of the medium has been adjusted to 6.8. This medium is extremely useful because
milk contains the sugar lactose, and casein, lactoalbumin, and lactoglobulin. The
last three are proteins. So we can detect fermentation and proteolysis.
 Fermentation of sugar will lead to a pink color change due to acid formation.
 The breakdown of proteins leads to ammonia formation which raises the pH and
leads to a blue color change.
 Litmus reduction leads to a white color change. This happens due to a drop in
oxygen and the dye is then used as an electron acceptor becoming reduced and
changing the medium to a white color.
 Coagulation of proteins can lead to curd formation.
 Peptonization or the medium becomes translucent or sometimes brown due to the
breakdown of milk proteins.
 Ropiness or the formation of thick slime due to the accumulation of waste
products and cells.
Procedure
1- Inoculate a litmus milk tube with your unknown.
Again, these are multiple test media that can detect physiological characteristics of
different bacteria. We exploit the fact that organisms behave differently to differentiate
them. Once we know what they are we can find an antibiotic to stop them.
From this point on we go by experiment and not week. Since these experiments were
multi step projects, I think its best to discuss each one individually!
Experiment 70; the identification and isolation of
Staphylococci
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The main focus believe it or not of this experiment was to isolate colonies of
Staphylococcus aureus. There are some properties you need to know
beforehand
1. Gram positive spherical bacteria that divide in more than one plain to
form irregular clusters.
2. When grown in trypticase soy agar or blood agar the colonies are 1 to
3 mm in diameter.
3. The colonies may be yellow, orange or white.
4. they are coagulase positive meaning they can clot serum
5. they produce alpha toxin to yield a clear zone of beta hemolysis in
blood agar plate
6. ferment mannitol sugar to produce acid
7. Are salt tolerant.
Know the chart of the three different species of Staphylococcus and their properties
S. aureus
S. epidermidis
S.saprophyticus
Alpha toxin
+
Mannitol (acid only) +
(+) mostly positive
Coagulase
+
Biotin for growth
+
Not significant
Novobiocin
Sensitive
sensitive
Resistant
To summarize each week
1st week
You inoculated three tubes of Staphylococcus broth with an unknown, a sample from
your nose and a sample from a fomite (object).
2nd week
You streak out two types of plates; MSA and SM110. The MSA or mannitol salt agar
contains mannitol sugar, phenol red indicator to allow us to detect mannitol fermentation,
and 7.5% NaCl since Staphylococci loves salt!
The SM110 also contains salt, mannitol but no indicator. The drawback is you can’t
see the yellow color change due to manniol fermentation. But it does allow for the
different color colonies to be seen.
3rd week
1. You examined your plates.
The MSA should have given you colonies that caused the medium around it to turn
yellow. Most of you guys had this.
The SM110 should have given you different color colonies but again maybe it didn’t.
2- Since Staphylococci are coagulase positive we inoculated serum with some of
our samples and placed them in the water bath for 2 hours. After 2 hours they
had solidified the serum.
3- Since Staphylococci can break down DNA with DNAase, we streaked out a
DNAase plate to examine next week.
4- A gram stain to confirm purity was optional.
5- A blood agar plate was streaked to detect beta hemolysis.
4th week.
1- The DNAase plate should show a clear zone. Ours was not very clear. But still it
should be clear.
2- Our blood agar plates are the final confirmation that we have S. aureus! The
should show beta hemolysis of blood cells!
Very important, based on the properties of Staphylococci, we can isolate them as
seen in this experiment. Please know the properties above!!!
Experiment 71; isolation and identification of Streptococci.
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The main objective here was to isolate streptococci and characterize them
based on certain properties. Pretty much you need to be able to identify them
according to Lancefield group, A, B, C, D or none. Know table 53.1
Know first 2 differences between the Staphylococci and Streptococci.
Week by week summary
Week 1
1- You collected Streptococci samples from your throat using a sterile
cotton swab and then swabbed and streaked a blood agar plate.
2- Since you were not given an unknown tube, we relied only on the throat
cultures! So in a way we had no positive control. We were hoping our
throat cultures would do the job!
Week 2
1- We looked for alpha or beta hemolysis. We as a class isolated only the
alpha hemolysis colonies. So this means that from now on our unknown
colonies can only be from group D or Streptococcus pneumoniae. What
is he saying? Well, look at table 71.1! Beta hemolysis is only found in
groups A and B streptococci. So we have knocked those two groups out!
2- Now we have to determine if our colonies are group D enteric, group D
nonenteric or oral viridians.
3- We picked isolated colonies and grew them in Trypticase soy broth.
4- Next week we will inoculate various media to determine what kind of
Streptococci we have isolated.
Week 3
1-So we streaked a blood agar plate and placed an optochin disk on the agar
surface.
2-We inoculated a bile esculin tube to detect esculin hydrolysis by group D
organisms.
3- We inoculated a 6.5% NaCl broth since all enteric group D species can
grow heavily here.
4- We inoculated a trypticase soy broth to determine bile solubility next
week.
Week 4 (Thank God!)
1- We examined the plate and determined that our organism was not susceptible to
Optochin. Optochin is an anti bacterial agent. Not an antibiotic!
2- We examined the Bile esculin slant. Group D organisms are positive for
esculin hydrolysis.
3- To differentiate between the enteric and nonenteric, we look at the 6.5% NaCl
tube. Enteric Streptococci are positive for 6.5% NaCl. Nonenteric can’t grow well
in 6.5 % NaCl.
4- If our species would have been susceptible to optochin, we then could have
checked for bile solubility. Streptococcus pneumoniae is optochin susceptible and
soluble in bile.
Experiment 72; Gram negative intestinal pathogens
1- Not much to say here but your aim was to isolate salmonella from two other
intestinal pathogens.
2- We grew the mixed culture of E. coli, P. vulgaris and Salmonella in 2 special
plates that will inhibit others including gram + bacteria. Do not forget, when one
goes to the doctor with intestinal problems, we get a sample that might contain
gram + bacteria. So we need to inhibit them. The media used were MacConkey
and Hektoen Enteric agar.
3- The following week, we isolated nice colonies from the two selective media. The
MacConkey colonies were nice and red and the ones in HE agar were nice and
green.
4- We then inoculated Russell double sugar slants.
5- These contain glucose and lactose. We are looking for colonies that only ferment
glucose. We need to pick only colonies form the tubes that will be half red and
half yellow. This would mean they only fermented the glucose. We are looking
for these only since salmonella doesn’t ferment lactose.
6- The third week, we took colonies from the tubes that showed only glucose
fermentation and inoculated SIM medium and Urea slant.
7- Salmonella is motile and should diffuse throughout the medium and it can’t
breakdown urea.
8- And finally this week, you take our final and quickly look at our tubes. You
should see diffusion in SIM tube and no reaction in the urea slant.
9- Also what has appeared on the final of past years is page 210, where you need to
know the different media used to differentiate between Shigella and Salmonella.
10- Mac Conkey agar, Salmonella and Shigella and other non-lactose fermenting
species produce smooth colorless colonies. Coliforms that ferment lactose
produce reddish or dark centered colonies.
11- Hektoen Enteric agar, Salmonella and Shigella colonies are greenish-blue. Some
species of Salmonella will have greenish-blue colonies with black centers due to
Hydrogen sulfide production. Coliform colonies are salmon to orange and may
have a bile precipitate.
12- Xylose Lysine Desoxycholate agar, most Salmonella colonies are red with a black
center; Shigella colonies are red. Coliform colonies are yellow.
13- REMEMBER, SHIGELLA IS NON-MOTILE AND WE CAN USE SIM TO
DIFFERENTIATE BETWEEN IT AND SALMONELLA, WHICH IS MOTILE!
Experiment 46, Enterotubes!



These tubes are really nice because they allow you to inoculate 12
different media and perform as many as 15 biochemical tests all at the
same time. It saves time and space and reagents.
You picked colonies form your experiment #72 plates and then after one
week you guys analyzed the results.
Make sure you can interpret the color changes. Look at TABLE 46.1 For
example, when bacterial decarboxylation of lysine leads to cadaverine
formation. Cadaverine is an alkalinic product that will result in a purple
color. So you will see a color change from yellow to purple for that
specific test.
Glucose /gas
Testing for
Glucose
fermentation
and gas
formation
Lysine
decarboxylation
Ability to break
down lysine
into Cadaverine
Ornithine
decarboxylation
Ability to break
down ornithine
into Putrescine.
Ability to
produce these 2
products
Hydrogen
sulfide/indole
production
Adonitol fermentation
Lactose fermentation
Arabinose
fermantation
Sorbitol fermentation
Voges- Proskauer
Ability to
ferment this
sugar
Ability to
ferment this
sugar
Ability to
ferment this
sugar
Ability to
ferment this
sugar
Ability to
undergo neutral
end product
fermentation
Dulcitol/Phenylalanine Ability to
deaminase
ferment Dulcitol
and the
presence of
phenylalanine
deaminase
enzyme
Color change
Red to yellow +
and a separation
in the wax
indicates
formation of
gas.
Yellow to
purple
+
Yellow to
purple
+
We look for a
black
precipitate and
add kovac’s
reagent to
detect the
indole ring
Red to yellow +
Red to yellow +
Red to yellow +
Red to yellow +
A red ring after
20 minutes wait
period. Add
barritt A and B
Red to yellow
indicates ability
to ferment the
sugar
After adding
10% iron
chloride the
explanation
We detect the
presence of
fermentation
by detecting a
drop of pH.
Cadaverine
raises the pH
and causes the
color change.
Raise of pH
causes color
change
No explanation
needed.
Drop in pH
causes color
change
Drop in pH
causes color
change
Drop in pH
causes color
change
Drop in pH
causes color
change
We detect the
production of
acetoin, a
byproduct of
the reaction
I have no
comments!
urease
Test for the
breakdown of
urea
Citrate fermentation
Test for ability
to ferment
citrate
presence of
pyruvic acid
should cause a
color change to
green/yellow.
Orange to pink
Green to blue
Raise in pH
due to
ammonia
buildup
Ammonia
buildup raises
pH and causes
color change
The end.
TRY THIS SHORT QUIZ TO TEST YOUR KNOWLEDGE OF EXERCISES 70, 71,
72, AND 46.
Please choose the best answer and fill in the scantron correctly.
1- Which species of Staphylococcus is resistant to Novobiocin?
a) S. aureus
b) S. epidermidis
c) S. saprophyticus
d) S. lugdunensis
2- True or False
To confirm the existence of S. aureus one should look for beta hemolysis on a blood agar
plate.
3- What indicator in the MSA medium allows us to differentiate S. aureus from the other
strains of Staph?
a) Phenol red indicator
b) Mannitol
c) Sodium chloride
d) Lactose
e) Litmus indicator
4- What enzyme present in S. aureus allows it to digest DNA?
a) Amylase
b) Catalase
c) DNase
d) RNase
5- What toxin present on S. aureus is responsible for beta hemolysis?
_________________
6- Identify the species of Streptococcus that exhibits beta hemolysis, is a group B, and is
negative for bile solubility. ______________________________
7- Identify the species of Streptococcus that exhibits alpha hemolysis, is negative for bile
esculin hydrolysis, and is not tolerant to 6.5% NaCl. ________________________
8- Identify the species of Streptococcus that exhibits beta hemolysis, is negative for the
CAMP reaction, and resistant to SXT. ______________________________
9- Identify the group D species that exhibits alpha hemolysis and is tolerant to 6.5% Salt.
_______________________.
10- What tests were performed to confirm the presence of an alpha hemolysis strain of
Strep?
_________________________, ______________________, __________________, and
____________________________.
11- In order to detect the CAMP factor, what organism besides your beta strain is
inoculated in your blood agar plate?
12- S. agalactiae is a group ________________ organism because it undergoes
__________________ hemolysis, is _______________ (sensitive or resistant) to SXT
antibiotic, and ______________ (positive or negative) for bile esculin hydrolysis.
13-How does a student differentiate between Streptococcus group D and Enterococcus
group D organisms?
14- What are 2 differences between Staph and Strep?
15- In MacConkey agar, Salmonella, Shigella and other non-lactose fermenting species
produce _________________ colonies.
a) Red
b) Pink
c) Colorless
d) Blue
16- In the Hektoen Enteric agar, Salmonella and Shigella produce colonies that are
_____________.
a) Greenish blue
b) Red
c) Black
d) Colorless
17- In the Xylose Lysine
Deoxycholate agar most ________________ produce
colonies that are red with a black center while Shigella colonies are _____________.
a) Salmonella, red
b) Streptococcus, black
c) Pseudomonas, red
d) Shigella, red
18- In the enterotube II system, the enzyme lysine decarboxylase removes a carbon
dioxide from lysine and forms _______________, an alkalinic end product. This end
product causes a color change from pale yellow to purple.
a) Indole ring
b) Pyruvate
c) Cadaverine
d) Glucose
19- All pathogenic species of Neisseria should be handled as soon as possible because
they are sensitive to environmental conditions. They are easily destroyed in samples that
a) Delayed in transit to the lab
b) Kept at a temp too far below or above 35 degree C
c) Heavily contaminated with normal flora
d) Not provided with enough carbon dioxide
e) All of the above
20- Gonorrhea, a sexually transmitted disease is caused by
a) Virus
b) Fungi
c) Bacterium
d) Algae
21- Zygospores are form by the union of nuclear material from the _____________ of
two different strains.
a) Hyphae
b) Spores
c) Nuclei
d) Cytoplasm
22- The only asexual fungi spore is known as a
a) Blastospore
b) Zygospore
c) Endospore
d) Gamete
We out!