Dr. Santos Microbiology lab notes 2018-2019Lab SafetyPlease 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 3Please go over

The compound light microscope and the parts of the microscope.Part of the microscopeA- 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.

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.

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.

Lab #2 exercises 9, 10 and 11Please 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.

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 16Please 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 fixStep 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.

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 Nocardia have a layer of mycolic acid that prevents them from being properly stained.

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 16Please 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 8Please 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. 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 1. carbon source2. nitrogen source3. vitamins 4. water5. energy source6. 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 eukaryote2. non-photosynthetic3. lack tissue differentiation4. have cell walls of chitin5. propagate by spores6. unicellular fungi are yeast7. 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 26Please go over

Please know the five groups of bacteria based on air requirement.1. obligate aerobe2. obligate anaerobe3. facultative anaerobe4. microaerophiles5. aerotolerant organisms

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 37Please 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 pathogenicityN. gonorrhoeae STD GonorrheaN. meningiditis meningitisN. sicca Normal flora of respiratory tractN. flavescens Normal flora of respiratory tract

• 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 laboratory2-Kept in temperature too far below or above 35°C3-Heavily contaminated with normal flora

4-Not provided with an adequate supply of carbon dioxide

The Spirochetes (27.2)Basic properties:

1- Slender, coiled organism2- Axial filament allows motion3- Thin cell wall4- 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 glucose2. specific sugars such as glucose, lactose and mannitol3. mixed acid fermentation4. butanediol fermentation5. 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 interpretationP. aeroginosa Green yellow oxidativeE. coli Yellow Yellow facultativeunknown 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 fermentation1. 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 test1. 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!

Oxidase1. 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- p- phylenediamine 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 test1. 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 reduction1. 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 acid4. reagent b is dimethyl –alpha- Napthylamine5. 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 43Please 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 detectedAmylase(B. subtilis)

Starch agar plate Add iodine look for clear zone due to starch degradation

We look for clear zone

Proteases(B.subtilis)

Skim milk plate Look for clear zone due to degradation of casein protein

Clear zone of degradation

Lipases(S.aureus)

Spirit blue agar We look for a dark blue precipitation due to lowering of pH as a result of fatty acids being released

Blue precipitation or sometimes just depletion of fat droplets on the agar.

Tryptophanase(E.coli)

Tryptone broth We add kovac’s reagent to detect the indole ring produced.

Tryptophan is broken down into indole, pyruvate and ammonia. We detect the indole.

Urease(P.vulgaris)

Urea agar slant When urea is broken down it releases ammonia and carbon dioxide. The ammonia raises the pH and the phenol red indicator causes a color change from yellow to bright pink

We detect the raise in pH due to the ammonia being released.

Phenylalanine deaminase

(P.vulgaris)

Phenylalanine agar We look for a greenish precipitate after the addition of 10% ferric chloride

Phenylalanine is broken down by the enzyme into 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!

Procedure1- 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.

Procedure1- 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 52; The identification and isolation of Staphylococci

The main focus 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 serum5. they produce alpha toxin to yield a clear zone of beta hemolysis in

blood agar plate6. ferment mannitol sugar to produce acid7. Are salt tolerant.

Know the chart of the three different species of Staphylococcus and their propertiesS. aureus S. epidermidis S.saprophyticus

Alpha toxin + - -Mannitol (acid only) + - (+) mostly positiveCoagulase + - -Biotin for growth - + Not significantNovobiocin Sensitive sensitive Resistant

To summarize each week1st weekYou inoculated three tubes of Staphylococcus broth with an unknown, a sample from your nose and a sample from a fomite (object).

2nd weekYou 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 week1. 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 53; Isolation and identification of Streptococci.

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 11- 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 21- 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 53.1! 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 31-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.

Summary of test to be done!

Experiment 54; 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!

Shigella/Salmonella

MacConkey Colorless

Hektoen Enteric Greenish/blue

XLD Shigella-redSalmonella- red/ black center

EMB Colorless

Experiment 41, EnteroPluri-Test System! 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 from your experiment #54 plates and then after one week you guys analyzed the results.

Make sure you can interpret the color changes. Look at TABLE 41.1 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.

Testing for Color change explanationGlucose /gas Glucose

fermentation Red to yellow + and a separation

We detect the presence of

and gas formation

in the wax indicates formation of gas.

fermentation by detecting a drop of pH.

Lysine decarboxylation

Ability to break down lysine into Cadaverine

Yellow to purple+

Cadaverine raises the pH and causes the color change.

Ornithine decarboxylation

Ability to break down ornithine into Putrescine.

Yellow to purple+

Raise of pH causes color change

Hydrogen sulfide/indole production

Ability to produce these 2 products

We look for a black precipitate and add kovac’s reagent to detect the indole ring

No explanation needed.

Adonitol fermentation Ability to ferment this sugar

Red to yellow + Drop in pH causes color change

Lactose fermentation Ability to ferment this sugar

Red to yellow + Drop in pH causes color change

Arabinose fermantation

Ability to ferment this sugar

Red to yellow + Drop in pH causes color change

Sorbitol fermentation Ability to ferment this sugar

Red to yellow + Drop in pH causes color change

Voges- Proskauer Ability to undergo neutral end product fermentation

A red ring after 20 minutes wait period. Add barritt A and B

We detect the production of acetoin, a byproduct of the reaction

Dulcitol/Phenylalanine deaminase

Ability to ferment Dulcitol and the presence of phenylalanine deaminase enzyme

Red to yellow indicates ability to ferment the sugarAfter adding 10% iron chloride the presence of pyruvic acid should cause a color change to

I have no comments!

green/yellow.urease Test for the

breakdown of urea

Orange to pink Raise in pH due to ammonia buildup

Citrate fermentation Test for ability to ferment citrate

Green to blue Ammonia buildup raises pH and causes color change

The end.