Unit 7 Microbiology: Inside and Out

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General and Medical Microbiology Lab ■ 199

Unit 7

Microbiology:

Inside and Out

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Lecture 20

Tasks for the day:

■ Cover Lecture 20 material. Vodcast found at: http://youtu.be/V3b7xDt8EAs ■ Perform Experiment 20A: Hemagglutination

■ Perform Experiment 20B: Ouchterlony Gel Diffusion, Lab Period One ■ Perform Experiment 20C: WBC Differential Blood Smear

I. Immunology: Types of defensive responses

A. Immunology is the study of ______________________ to ________________________________ _______________________________ (e.g. bacteria, viruses, and non-self proteins)

B. Defense mechanisms 1. Innate mechanisms

a. Develop ______________________ and deal with invaders using ___________________________________________.

b. Weapons include:

i. Physical barriers like skin and mucous membranes ii. Secreted enzymes

iii. __________________________ factors

iv. ______________________________________ 2. _______________________ mechanisms

a. Develop ___________________________________________ in response to the foreign agent (antigen or immunogen). _______________________________________ against the antigen. b. Have memory, so they are _______________________________________ the antigen is encountered.

c. Depend primarily on specialized cells: __________________________________________ ____________________________.

C. Specific immunity

1. Humoral immune response

a. The presence of an antigen stimulates ______________ to produce __________________ (glycoproteins that react specifically with the antigen and target these antigens for

_______________________________). 2. _______________________ immune response

a. _____________________________________ and attack cancerous cells, transplanted cells, or host cells that have been ______________________ by viruses or microorganisms.

II. Antibodies and antigens

A. Antibodies (Ab) are ______________________________ (have two equally specific binding sites for the same antigen); ______________________________ with the following structure:

1. __________________________

a. Differences in this region account for antibody specificity 2. _________________________

3. _________________________

a. Antibodies in the same class have virtually identical amino acid sequences in this region. 4. ____________________________________________

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General and Medical Microbiology Lab ■ 201 a. These are lacking in IgB and IgM.

B. Antigens (Ag) are

______________________. Each antigen has many ____________________________ (antigen _______________________ that generally consist of 5-10 amino acid or sugar residues).

III. Antigens and antibodies interact with one another in detectable ways.

______________________________ is the science of

___________________________________ _________________________ these antigen-antibody reactions. A. There are two types of antigen/ antibody reactions

1. Because antibodies are bivalent, they can react with two antigen molecules or cells of the same type. This allows them to ________________________________ _________________________________ _________________________________ _____________________________ of alternating Ag-Ab-Ag-Ab-etc.

2. Eventually, this antigen-antibody lattice network becomes so large that

it_____________________________ __________. If the antigen is a

_______________________ antigen, such as a protein or a carbohydrate, this reaction is called a

______________________________ ____________. If, instead, the antigen is ______________________, such as a red blood cell or bacteria, the reaction is called an _______________________. How Does It Work? Anitbodies Antigens (Ag) Antibody/Antigen Lattice Network

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General and Medical Microbiology Lab ■ 202 B. There are two kinds of experiments to demonstrate the two types of antigen-antibody

interactions.

1. Agglutination (__________________________________), which includes blood typing for ABO and Rh factor

a. ABO blood typing

i. Mix a drop of blood with Antibody-A, Antibody-B, and Antibody-Rh factor in separate wells. Hemagglutination in the wells indicates that the _________________

_____________________________________________________________________.

In vivo, people produce antibodies to surface glycoproteins they see as foreign. Blood Type Antibody produced Ability to accept/donate

A Antibody-_________________ Can accept type _____________

B Antibody-_________________ Can accept type _____________

AB _________________________ Universal _______________

O Antibody-_________________ Can accept only type ____________; Universal _______________

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General and Medical Microbiology Lab ■ 203 b. Rh factor:

i. Based on the presence or absence of the RhD antigen on RBCs. An individual who has a blood type of A and is RhD+ is said to have ___________ blood.

ii. RhD- people must be _______________________________ to the RhD antigen before their body will start making antibodies.

iii. The Rh factor is important to an Rh- woman who is carrying a child of an Rh+ man.

______________________________________________________. The woman can produce antibodies to the Rh factor and, during subsequent pregnancies, the woman’s immune system treats the presence of the Rh factor as an infection and effectively neutralizes it, resulting in abortion. This is called “hemolytic disease of the newborn” or ________________. To prevent this, there is a therapy available called __________________. This therapy effectively gives the mother bunches of antibodies after the first pregnancy to

_____________________________________.

2. Precipitation, which includes observing the interaction between a protein antigen a. _______________________________

b. This test will be done on an agar gel to immobilize the precipitates so they are more easily observed.

c. Using this test, we can determine ________________________________ and still give positive precipitation.

i. If the last positive precipitation band is seen for the 1/8 dilution, then the antigen can be diluted 8-fold and still give a positive precipitation reaction. In immunology, this dilution factor is often referred to as the ____________________, even though it is just a unitless factor.

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General and Medical Microbiology Lab ■ 204 ii. FYI: Ouchterlony gel precipitations have been used in the

understanding of how

______________________________________

____________________________ interact with the immune system— women make antibodies to them.

IV. Wright stained blood smears

A. Never Let Monkeys Eat Bananas: What?

1. This is a mnemonic designed to help you remember the names of white blood cells in order of decreasing abundance.

a. Never

i. _________________________________ are the most abundant type of white blood cells in our serum. They function mainly in the nonspecific immune response in a __________________________ manner. They are classified as _____________________________, which means their cytoplasm is filled with granules (orlysosomes).

b. Let

i. _______________________________ are agranulocytes — free of granules — that function in the

_________________________________ (cell-mediated and humoral).

c. Monkeys

i. ______________________________ are eventually phagocytic when they mature into

___________________________________. They are

predominately involved in the nonspecific immune response and are classified as agranulocytes.

d. Eat

i. ________________________________ are hardly present in our blood and function during

________________________________ in a phagocytic manner. They areclassified as granulocytes.

e. Bananas

i. _________________________________ are very few and far between. They function during allergic reactions to release heparin and histamine. These cells work in concert with eosinophils. They are classified as granulocytes.

B. Red Blood Cells

1. These are the largest population of blood cells and function to transport oxygen and carbon dioxide. They are a

_______________________________ shape which increases the surface area to volume ratio for effective

_______________________________________________. Eosinophils Neutrophils Leukocytes Monocytes Basophils

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Experiment 20A:

Hemagglutination

In today’s lab period, we will determine the ABO and Rh blood type of two different samples using a simulated human blood-typing kit.

Note: The blood in this simulated blood-typing kit is not actual blood. Thus, we need not be concernedabout blood borne pathogens such as HIV or hepatitis B. However, when working with actual blood it is very important to use extreme care to avoid contamination. In order to be prepared for future experience, we will perform this procedure as if we were working with actual blood. Wear disposable gloves, a lab coat, and safety glasses at all times. Place all contaminated material in a biohazard bag.

Procedure

Students will work in pairs.

The blood-typing station is set up on the back bench.

1. Place 1 drop of Antiserum A into the well labeled “A.” 2. Place 1 drop of Antiserum B into the well labeled “B.”

3. Place 1 drop of Antiserum D into the well labeled “D” or “Rh.”

4. Choose to work with the virtual blood of one patient. Place 1 drop of blood in each of these three wells of the blood-typing slide. Be careful not to allow the dropper to touch the antiserum.

5. Using a sterile toothpick for each well, thoroughly mix the blood and antiserum. Touch the mixture to the sides of the well to break the surface tension.

6. Place the slide on the light box and gently rock it back and forth for about 1 minute. If the mixture appears to be uniform, there is no agglutination (-). If the mixture appears granular, there is agglutination (+).

7. Repeat the above procedure for a second patient. 8. Discard all materials in the biohazard waste container.

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Experiment 20A:

Results

1. Record the blood typing results in the table below.

Unknown Sample Antiserum A* Antiserum B* Antiserum D* Blood Type **

Patient #________ Patient #________

Indicate positive or negative hemagglutination reaction for each antiserum From the hemagglutination

2. Can the first patient tested receive blood (without ill effects) from the second? Explain.

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 3. Can the second patient tested receive blood (without ill effects) from the first? Explain.

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 4. Why is blood type O/Rh- (O-) considered the “universal donor”?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 5. Why would a person with blood type A exhibit a transfusion reaction if given type B blood?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 6. What is hemolytic disease in the newborn? How does RhoGam prevent it?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 7. Describe how the agglutination, or clumping, occurs in the blood-typing procedure.

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

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Experiment 20B:

Ouchterlony Gel Diffusion

This is an example of a precipitation reaction between antibodies and soluble antigens — in this case, proteins. The bivalent antibodies will cross-link the soluble antigen to form a lattice network and yield a visible precipitate. As with any antibody-antigen reaction, the antibody must specifically recognize the antigen. In this experiment, bovine serum albumin, or BSA, will be used as the soluble antigen and anti-BSA antiserum as the antibody preparation. This procedure permits quantitative and qualitative characterizations of the antibody/antigen interaction.

Lab Period One

Procedure

Students will work in groups of 4.

From the side bench, collect 1 tube containing BSA, the antigen, diluted 1:2; 1 tube containing (alpha)-BSA, the antibody to BSA; 1 tube containing sterile saline; 6 Eppendorf, also known as eppi or microcentrifuge, tubes.

1. The Ouchterlony plates are already poured and the wells have been punched out. In order to suck the plugs from the wells, attach a Pasteur pipet to a vacuum flask. Carefully suck the plugs from the wells.

Take caution to only remove agarose from the wells and not from the surrounding area.

2. Prepare a serial dilution of BSA. Label the 6 Eppendorf tubes #1 through #6.

a. Dispense 50 μL of saline (0.85% NaCl) into each of the 6 tubes labeled #1 through #6. Use a P200 pipetman. It is not necessary to change pipet tips between tubes, as the solution is being dispensed into sterile, empty microcentrifuge tubes.

b. Transfer 50 μL of BSA antigen to tube #1. Draw the solution up and down gently 4-5 times in order to mix the solution and rinse the pipet. Do this gently to avoid making bubbles!

c. Using a new pipet tip, transfer 50 μL from tube #1 to tube #2. Mix as in step b.

d. Using another new pipet, transfer 50 μL from tube #2 to tube #3. Mix. Proceed the same way until the last tube is mixed. Discard pipet tips appropriately.

e. This series of two-fold dilutions will generate the following:

Tube: #1 #2 #3 #4 #5 #6

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General and Medical Microbiology Lab ■ 208 3. Label the bottom of an Ouchterlony plate. Follow the diagram below.

4. With a P20 pipetman, dispense 10 μL of the diluted antigen into the outer wells as labeled. If you start with the 1/128 dilution and continue to the ¼ dilution, you can use the same tip for all the antibody samples. 5. With a clean pipet tip, fill the center well with 10 μL of antibody (alpha)-BSA.

6. Be careful not to disturb the fluid in the wells. Incubate without inverting for 48 hours at room temperature in a humidified chamber (Tupperware with moistened paper towels in the bottom).

Lab Period Two

Procedure

1. Collect the Ouchterlony plates and observe the white lines between the wells containing antigen and antibody. These will be difficult to see and thus are most easily observed by holding the plate up to the light. Sketch these observations in the results section for this experiment.

Ag=Bovine Serum Albumin (BSA) Ab=antibody to BSA (a-BSA)

Ab Ag 1/4 1/8 1/16 1/32 1/64 1/128

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Experiment 20B:

Results

1. Sketch the results

2. What was the antigen in this test?

______________________________________________________________________________________ ______________________________________________________________________________________ 3. What was the antibody in this test?

______________________________________________________________________________________ ______________________________________________________________________________________ 4. Were the antigen and antibody tested specific for one another? How can we tell?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 5. By how much can the antibody be diluted and still allow for a visible reaction to occur?

______________________________________________________________________________________ ______________________________________________________________________________________ 6. What antibody dilution factor shows the optimal reaction?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 7. Is the Ouchterlony gel diffusion test an example of agglutination or precipitation? Explain.

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

Ag=Bovine Serum Albumin (BSA) Ab=antibody to BSA (a-BSA)

Ab Ag 1/4 1/8 1/16 1/32 1/64 1/128

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Experiment 20C:

WBC Differential Blood Smear

Depending on the condition of the patient at the time of the blood draw — and luck with a microscope — uncommon white blood cells, such as the blue- and purple-stained eosinophil above, could be observed in a smear.

During many kinds of bacterial infections, there is an increase in the total number of white blood cells, or WBCs. The increase is called leukocytosis. A good way to remember the relative proportions of WBCs is Never Let Monkeys Eat Bananas. Please note that, in addition to cells, one might also note small “cell-like”

constituents called platelets. These small constituents are important for clotting. Procedure

Students will work individually.

1. Examine prepared blood smears using the 10X and the oil-immersion lens. Attempt to find each type of WBC. Refer to the pictures provided in lab lecture on page 204 as a guide for identifying the different WBCs.

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Experiment 20C:

Results

1. Diagram each type of WBC observed at 1000X total magnidication

2. Which type of WBC is found most frequently?

_____________________________________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ 3. Are there any WBC types that were not found in the blood smears? If so, which one(s)? Why couldn’t they be found?

_____________________________________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________ 4. In what circumstance would eosinophil and basophile levels be elevated?

_____________________________________________________________________________________ _____________________________________________________________________________________ _____________________________________________________________________________________

Neutrophil Lymphocyte Monocyte

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Lecture 21

Tasks for the day:

■ Cover Lecture 21 material. Vodcast found at: https://youtu.be/K47--VPiX0Q ■ Perform Experiment 20B: Ouchterlony Gel Diffusion, Lab Period Two ■ Perform Experiment 21A: Water Microbiology, Lab Period One

I. Introduction

A. Water microbiology is the __________________________________________________. This is done to determine what organisms are present and if they are helpful or harmful.

B. The safety of water for drink and contact is the main reason for

________________________________________________________. We must know if the water will cause harm to us or the environment. Some microorganisms can actually reduce the amount of environmental damage. We treat drinking water to make it potable.

II. Harmful organisms (bacteria, parasites, and viruses)

A. ___________________________________________________ may affect as few as 1 or 2 people, or they may affect enough people to reach epidemic proportions. Luckily, the Safe Water Drinking Act and the

_______________________________ help keep us safe. Water treatment is not without flaws, so it is still a _______________________________ that drinking water is safe, especially with the increased number of HIV/AIDS patients.

B. Water contamination from _____________________________________________ introduces into the water:

1. Pathogenic bacteria (mostly enteric or GI tract) i. Vibrio cholera, which causes cholera

_____________________, including b. Salmonella typhi

ii. Cyanobacteria, which may be a risk factor in cancer

iii. Mycobacterium

2. ____________________________, including the “super bugs” Cryptosporidium and Giardia 3. Viruses, including hepatitis viruses III. Screening for pathogens

A. It is _______________________________ to screen for enteric pathogens (e.g. Shigella and _______________________) for several reasons:

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General and Medical Microbiology Lab ■ 213 1. These pathogens enter the water supply ______________________________ because not everyone is infected and shedding them continuously.

2. Most enteric pathogens are not stable in the environment, therefore they _________________________________________________.

3. These pathogens are generally ______________________________________________ so they may _____________________________________________.

4. _______________________________________________ are required to culture enteric pathogens.

B. Due to these problems associated with finding pathogens in large volumes of water, we

will instead look for _______________________________________________ whose presence indicates fecal contamination. The most common group of these indicator organisms are called

___________________________.

C. Coliforms are __________________________________ that are present in the

___________________________ of every warm-blooded animal. They are defined as aerobic or facultative anaerobic Gram-negative rods that do not form spores, but do

_____________________________________________________. The

____________________________________________ than enteric pathogens for several reasons:

1. Animals and humans __________________________________________________ in their GI tract.2. They are _________________________ in feces

3. Their presence in water will indicate fecal contamination, which suggests the possibility of pathogen contamination.

4. Coliforms ___________________________________ in water and are much ________________________________________ in the lab.

D. The detection of coliforms is complicated by the fact that _____________________________________

________________. In fact, some are commonly found in the environment. Because of this, we commonly distinguish coliforms biochemically, to determine

____________________________________ using the ____________________________________________.

IMViC: ______________________ _____________________________

Organism Indole MR VP Citrate

Fecal coliforms (Escherichia coli) Non-fecal coliforms (Enterobacter aerogenes)

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General and Medical Microbiology Lab ■ 214 IV. We will screen wastewater treatment plan (WWTP) influent and effluent, Laramie River water, water

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General and Medical Microbiology Lab ■ 215 V. Tests for coliforms

A. The ______________________________________

1. The PA is used to quickly examine for the presence of coliforms. This test is able to detect _________________________________________.

2. The PA broth contains __________________ and a pH indicator that will change from a purple color to a _________________________________ from the fermentation of lactose.

B. The ____________________________ ___________________ is a test that may be used to assay a

________________________________ ________ and allow us to

_______________________________ the number of coliforms in that sample.(See How Does It Work, right)

1. The water is passed through a 0.45 micron

_____________________________. 2. The filter is then placed on a selective and differential medium, usually EMB or Endo agar, which allows for identification of coliforms.

i. Endo media contains sodium sulfite and basic fuchsin to inhibit the growth of _______________________ organisms. Coliforms that ferment lactose form red or pink colonies that may be ___________________________.

Knowledge check Which water sample

contains at least 1 coliform?

How Does It Work? Membrane Filtration

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General and Medical Microbiology Lab ■ 216 D. The Multiple Tube Fermentation Method is a test that is performed in three stages.

1.____________________________________ uses a ______________________________ that will be used to determine the presence of lactose fermenting bacteria in the sample. The nine inoculated lactose broth tubes will also enable us to ___________________________________ the amount of coliforms present in our sample by using the most probably number, or MPN, table located on page 220.

Profile: ______________________________

How many coliforms/100 mL are present in this sample?

2. ____________________________________ is performed by inoculating MacConkey agar that contains MUG (4-Methylumbelliferyl--D-glucuronide). Selected Gram-negative colonies that ferment lactose turn bright pink; if the enzyme ____________________ is present, it will hydrolyze MUG to form a compound that __________________ under long-wavelength UV. Because 96-97% of E. coli strains produce -glucuronidase, this test is said to verify E. coli colonies.1

3. ________________________________________ is done by selecting a lac+, fluorescent colony from the MacConkey/MUG plate and __________________________________________ to verify lactose

fermentation. Also, the same colony should be stained to verify the Gram-negative, _________________________________ character.

E. These tests are done on numerous bodies and sources of water

________________________________________________________________________. VI. Other microbiology applications in water treatment

A. Biosensing is one of the new breakthrough in microbiology. Korean scientists are using

___________________________ containing the lux operon to indicate if there has been a failure in wastewater treatment.

B. Britain has the Microtox System, which uses Photobacterium directly to detect pollutants. They can’t ___ _____________________________________________________________________.

C. Pseudomonas expressing _____________________________ and other genes encoding toluene or benzene recognition will fluoresce in presence of these pollutants.

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Experiment 21A:

Water Microbiology

Bacteriologic testing of water is done to ensure that enteric pathogens, capable of causing infectious disease in people consuming the water, are not present. During this experiment, we will be testing samples of water for the presence of coliforms. Each table will receive a water sample from a different water source, including the Laramie River, Laramie Water and Wastewater Treatment Plants, the tap, etc. Record the source of water sample that your group is analyzing. We will use three methods to screen the water samples: P-A (Presence-Absence) test; membrane filtration technique; and the multiple Tube Fermentation Method (MTFM). If these methods used to screen the water can detect no coliforms, then the water is considered safe to drink, also called “potable.”

Lab Period One

A. P-A (Presence-Absence) test

P-A broth contains lactose and a pH indicator, which will change from purple to yellow if lactose is fermented and acid is produced.

Procedure

1. Using a sterile graduated cylinder, add 100 mL of the appropriate water sample to 50 mL of 3X P-A broth. 2. Tighten the cap and invert the bottle 5-6 times to mix. Loosen the cap ¼ of a turn to allow O2 to enter the bottle during incubation.

3. Place the bottle in the tray on the side bench to be incubated for 48 hours at 37°C.

B. Membrane Filtration technique

The membrane filtration method offers some advantages over other methods because it allows a much larger volume of water (for example, 100 mL) to be tested. Therefore, it might detect lower levels of contamination. It is also a quantitative test that determines the actual number of coliforms present per 100 mL volume. Water is filtered through a membrane with 0.45 mm pores. This membrane filter is then placed on a pad saturated with m-Endo MF broth, a selective and differential medium. After a 24-hour incubation period at 37°C, colonies of coliforms, if present, will be dark pink/red and might exhibit a metallic sheen.

Procedure

Students will work in groups of 4

Each group of 4 will filter 3 different volumes of their water sample: 1 mL, 10 mL, and 100 mL.

1. Label the sidewalls of the 3 Petri dishes with your initials, lab section number, water sample source, and volume of water filtered (1 mL, 10 mL, and 100 mL).

2. At the m-Endo broth station on the side bench, collect 3 small Petri dishes. Place a sterile pad into the bottom of each dish and saturate each pad with 2 mL of m-Endo broth.

3. To optimize filtration, it is important to have a total volume of at least 100 mL. Therefore, it is necessary to add the 1 mL and 10 mL samples to 100 mL water blanks as described below:

a. Collect two 100 mL water blanks and label as 1mL and 10 mL. b. Pipette 1 mL of the water sample into the first 100 mL water blank. c. Pipette 10 mL of the water sample into the second 100 mL water blank.

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General and Medical Microbiology Lab ■ 218 4. For the third sample, simply measure 100 mL of the water directly into the 100 mL graduated cylinder.

a. At this point, every group should have 3 water samples — 2 diluted and 1 undiluted. All samples should be approximately 100 mL in volume.

5. Follow the procedure below to perform the filtrations:

a. Collect three membrane filters with grids. Assemble the filtration unit as follows: i. Insert the funnel base into the side-arm flask

ii. Using flamed forceps, aseptically place a membrane filter (grid-side up) on the screen support. Be certain that only the filter, which is white in color, is placed on the screen. The filter is packaged between two blue sheets of paper, which, if placed on the grid, will stop all water from passing through the filter.

iii. Place the funnel securely on the base. iv. Be sure all connections are firm! b. Attach rubber tubing to the vacuum line.

c. Vigorously shake the water bottle containing the 1 mL sample (diluted to 100 mL in water) to disperse the bacteria. Pour the sample into the funnel. Slowly turn on the vacuum.

d. Without removing the filter, rinse the inside of the funnel with about 50 mL of sterile water. Turn the vacuum off. Release the vacuum by disconnecting the tubing.

e. Remove the funnel and set it on the foil wrapper. It will be used for the next sample.

f. With flamed forceps, transfer the filter to the labeled dish. Keep the grid side up. Gently push the filter down onto the media-saturated pad so there are no air bubbles.

g. Repeat the procedure for the 10 mL and 100 mL samples.

5. Incubate the plates with the lids up (do not invert the plates) in a humidified chamber at 37°C.

6. Place all used equipment on the designated tray. Pour the filtrate in the vacuum flask down the sink. This should be really clean water! Label the flask and leave it on the bench for the next class.

C. Multiple Tube Fermentation Method (MTFM)

This method is comprised of three stages:

1. The presumptive test, which we will do today

2. The confirmed test, which we will do during Lab Period Two. 3. The completed test, which we will do during Lab Period Three.

In the presumptive test, 9 different tubes of lactose broth with 10-fold variations of the water sample are

inoculated. If any lactose-fermenting bacteria grow and produce gas, the medium will turn yellow and bubbles will be present in the Durham tube. It is thus “presumed” that coliforms are present. This test is also used to determine the most probably number, or MPN, of coliforms present per 100 mL of water. The samples should be incubated for 48 hours at 37°C. If the test is positive, the confirmed test is performed.

Procedure

Students will work in groups of 4

From the side bench, collect 3 tubes of double-strength lactose broth, also known as DSLB; and 6 tubes of single-strength lactose broth, also known as SSLB.

1. Mix the water sample well.

2. Inoculate the 3 DSLB tubes with 10 mL of the water sample. Label these tubes with your initials, section number, water sample identity, and volume of the water used (10 mL).

3. Inoculate 3 SSLB tubes with 1 mL of the water sample. Label these tubes with your initials, section number, water sample identity, and volume of the water used (1 mL).

4. Inoculate 3 SSLB tubes with 0.1 mL of the water sample. Label these tubes with your initials, section number, water sample identity, and volume of the water used (0.1 mL).

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Lab Period Two

A. P-A (Presence-Absence) test

Procedure

1. Collect the P-A broth and record the results for lactose fermentation in the results section of this experiment.

B. Membrane Filtration technique

Procedure

1. Collect the membrane filters and count the number of colonies. Record the data in the results section of this experiment.

In this lab period, we will continue MTFM for water testing (presumptive, confirmed, and completed tests). Today, we will perform the confirmed stage.

Procedure

From the side bench, collect 1 MacConkey agar that contains MUG (4-methylumbelliferyl-β-D-glucuronide.

1. Collect your MTFM tubes and count the number of lactose-fermentation-positive tubes for each set of three (each volume

— 10 mL, 1 mL, and 0.1 mL — represents a set). Record the resulting profile in the results section of this

experiment and use the Most Probably Number table to determine the MPN of coliforms per 100 mL.

Remember: Apositive tube can be recognized by its yellow color.

2. Using an inoculating loop, obtain a sample of broth from any positive MTFM tube and T-streak for isolation onto a MacConkey agar that contains MUG

(4-methylumbelliferyl-β-D-glucuronide). Recall that

MacConkey agar is a selective and differential medium. It selects for Gram-negative bacteria by suppressing the growth of Gram-positive bacteria. It is differential because lactose fermenters form colonies that are bright pink in color (typical for Escherichia coli); the colonies of lactose non-fermenters appear uncolored or are similar in color to the medium. The addition of MUG to this medium enables the sensitive detection of E. coli. The MUG compound is initially colorless, but it can be hydrolyzed by the E. coli β-glucuronidase enzyme to form a compound (4-methylumbeliferone) that fluoresces under long-UV light (336 nm). Observing for fluorescence emission is a sensitive way to confirm the presence of E. coli in water samples1_.

1. American Society for Microbiology, Microbe Library (2009). Retrieved from http://www.microbelibrary.org/component/resource/ laboratory-test/3201-use-of-ec-mug-media-to-confirm-escherichia-coli-contamination-inwater-samples-protocol

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Lab Period Three

In this lab period, we will continue MTFM for water testing (presumptive, confirmed, and completed tests). Today, we will perform the completed stage.

Procedure

From the side bench, collect 1 lactose broth tube; 1 SIM tube; 1 MR-VP tube; and 1 Simmon’s citrate tube.

1. Collect your MacConkey plus MUG (4-Methylumbelliferyl--D-glucuronide) plates from the confirmed stage of the MTFM and record your observations in the results section of this experiment. Lactose-fermenting bacteria produce acidic byproducts that cause colonies to turn bright pink. Your TA or instructor will assist you in exposing the plate to UV light. E. coli, the most notable fecal coliform representative, colonies will fluoresce.

Note: Not all coliforms are of fecal origin; nonfecal coliforms likeEnterobacter aerogenesare also commonlyfound in the environment. Thus, coliforms may or may not signify fecal contamination of waters. If coliforms are detected by using one or more of the tests that have been performed (membrane filtration, P-A, MTFM), one can determine using the IMViC tests whether they are fecal or nonfecal coliforms.

Organism Indole MR VP Citrate

Fecal coliforms

(Escherichia coli)

+

–

Non-fecal coliforms (Enterobacter

aerogenes)

–

+

2. Using a sterile inoculating loop, pick up about half of a colony of the MacConkey plus MUG plate. Select a lactose-positive, fluorescent colony and inoculate a lactose broth tube to verify acid and gas production.

3. Using a sterile inoculating loop, select a single isolated, lactose-fermenting colony from your MacConkey plus MUG plate. This colony can be either fluorescent or non-fluorescent.; please feel free to choose one of either type. Inoculate the media for the IMViC tests — 1 SIM tube, 1 MR-VP tube, and 1 Simmon's citrate tube. Review the procedures for these tests in the procedures for Experiment 28: Unknown Identification.

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Experiment 21A:

Results

Source of Water Sample: _________________________________________________ A. P-A test

1. Was the test positive for lactose fermentation?

______________________________________________________________________________________ ______________________________________________________________________________________ 2. What follow-up tests could be done to confirm the positive test?

______________________________________________________________________________________ ______________________________________________________________________________________ 3. Which of the follow-up tests would give a quantitative measurement of the number of coliforms present per 100 mL?

______________________________________________________________________________________ ______________________________________________________________________________________ 4. Speculate as to why the P-A test is the procedure used by labs for routine inspection of drinking water. ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

B. Membrane Filtration Technique

1. Record the number of coliform colonies present on each filter, if countable

Some water samples might have been diluted prior to testing to yield optimum results. If so, this information will be provided by the TA. If the sample was diluted, the dilution will have to be included in the calculation of coliforms/100 mL. If your water sample was not diluted, disregard this column.

Coliform counts are commonly expressed per 100 mL water sample. Therefore, if the 1 mL sample gave a countable number of colonies, multiply the number of colonies by 100 to get coliforms/100 mL. If the 10 mL sample gave a countable number, multiply the number of colonies by 10 to get coliforms/100 mL.

Volume of water

sample filtered Dilution factor* Number of colonies Coliforms/100mL**

1 mL 10 mL 100 mL

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Presumptive test:

1. A set of 9 lactose broth tubes was inoculated with measured volumes of the water sample (3 with 10 mL; 3 with 1 mL; and 3 with 0.1 mL). Record the number of positive tubes for each volume.

For each volume, indicate the number of tubes (out of 3) that gave a positive lactose fermentation reaction. This is referred to as the “profile” of your water sample.

2. Using the table provided, determine the Most Probable Number (MPN) index for the water sample. MPN: ___________________ (This is per 100 mL sample.)

Class Data for the Membrane Filtration and Multiple Tube Fermentation Methods 3. Compile the class data for the various water samples that were tested:

Source of water sample Coliforms/100 mL (MTFM) Coliforms/100 mL (membrane filtration)

Number of tubes giving positive reaction*

Source of water 10 mL 1.0 mL 0.1 mL

Most Probable Number Table (adapted from Standard Methods of Water and Wastewater,

Twelfth edition) PROFILE Number of positives in the set of three 10 mL tubes Number of positives in the set of three 1.0 mL tubes Number of positives in the set of three 0.10 mL tubes MPN (Coliforms per 100 mL) 0 0 1 3 0 1 0 3 1 0 0 4 1 0 1 7 1 1 0 7 1 1 1 11 1 2 0 11 2 0 0 9 2 0 1 14 2 1 0 15 2 1 1 20 2 2 0 21 2 2 1 28 3 0 0 23 3 0 1 39 3 0 2 64 3 1 0 43 3 1 1 75 3 1 2 120 3 2 0 93 3 2 1 150 3 2 2 210 3 3 0 240 3 3 1 460 3 3 2 1100

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General and Medical Microbiology Lab ■ 223 4. Does the MPN agree with the results obtained from the membrane filtration method?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 5. Compare the membrane filtration method and the MTFM. Which method is the most accurate? Explain. ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ Confirmed Test:

1. A MacConkey with MUG agar plate was streaked with a sample from a positive lactose MTFM tube. Describe the appearance of the agar plate after it was incubated both with and without exposure to long-wavelength UV.

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 2. Did the MacConkey with MUG agar plate indicate the presence of coliforms?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ Completed Test:

1. Describe the results of the completed test.

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 2. Record the results of the IMViC test in the table below

Water Sample Indole MR VP Citrate

3. Based on the IMViC profile, is the isolate tested a fecal or non-fecal coliform?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 4. Based on the results of the MTFM test (presumptive, confirmed, and completed), is the water sample

potable? Explain.

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

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Lecture 22

Tasks for the day:

Cover Lecture 22 material. Vodcast found at: https://youtu.be/Eew9XX1BUcs Perform Experiment 21A: Water Microbiology, Lab Period Two

Perform Experiment 22A: Enumeration of Microorganisms in Soil, Lab Period One Perform Experiment 22B: Enrichment of Azotobacter, Lab Period One

I. Soil microorganisms are important in: A. Biogeochemical cycles

1. They ____________________________, and ___________________________________, such as _________________________________________, and sulfur. Ecosystem would collapse without soil microorganisms _______________________________.

B. Biopesticides

1. Proteins produced by some bacteria have been used to

_________________________________________. A toxin produced by Bacillus thurigiensis is so useful that the gene that encodes for the toxin was isolated from the bacteria and

_________________________________________________________. These genetically transformed _____________________________ and _____________________________ plants are now able to produce the ____________________________________ and protect themselves against damage by

_________________________________, such as moths and worms. B. thuringiensis has also been used to control _________________________________ and ___________________________________.

The 5 major components of soil A. Inorganic matter B. ___________________ C. _________________ matter (decaying biomass) D. Air E.The ______________________: Animals, plants, microorganisms, bacteria, archaea, fungi, ___________________________

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General and Medical Microbiology Lab ■ 225 C. Sources of antibiotics

Note: You are already familiar with the use of soil microorganisms as sources of severalantibiotics! II. Enumeration

A. The accurate enumeration of microorganisms in the soil is difficult because culture methods reveal only those few microorganisms that can grow in the _______________________________________

__________________________________. Direct microscopic examination of soil is also difficult and would not reveal virus particles or differentiate ____________________________________________.

B. We will attempt to isolate three groups of soil microrganisms: 1. ________________________

a. _________________________________ in the group Actinomycetes.

b. _________________________________ with hyphae, conidia and spores

c. Produce _______________________ which gives the soil an earthy odor

d. Source of several _____________________ (e.g. Streptomyces griseus produces streptomycin)

e. ____________________ medium is used for enrichment

2. _________________________

a. _______________________ of soil microbes have been cultured and characterized1.

b. Generally, there are more ______________________ bacteria (e.g. Agrobacterium, Cellulomonas, and Pseudomonas), but certainly Gram-positives, such as

_______________________ are present. c. Contamination may introduce

___________________________________ but, generally, these are rapidly eliminated.

d. ______________________ is used for enrichment 3. ___________________________

a. Abundant in _________________________ soils b. _________________________ that secrete enzymes to degrade polysaccharides, such as lignin and cellulose

c. Free-living species are more easily cultured than are those _________________________ species.

d. _________________________ is used for enrichment (a high carbohydrate content favors mold growth and streptomycin and rose bengal dye select against bacteria)

1. Statistic from Prescott, Harley, and Knein’s Microbiology (2008)

Streptomyces

Other bacteria

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General and Medical Microbiology Lab ■ 226 C. ________________________________

1. The plant cover in the soil is an important factor in determining the types and numbers of microorganisms in that soil.

___________________________ is the zone of soil that adheres to plant roots and is enriched in nutrients. Plant root exudates and senescent parts of plant excrete organic molecules, including

____________________________________, and _____________________________, and can be an important source of nutrients for soil microorganisms. At the rhizosphere, there are ___________________________________ than in the surrounding soil. The rhizosphere also enhances

_______________________________________. III. Nitrogen fixation

A. The ____________________________________ is one of the most important biogeochemical cycles with which microorganisms are involved.

1. Nitrogen is an essential building block for all amino acids and many other macromolecules.

Unfortunately, most nitrogen exists as N2 gas, which is not usable by most organisms. Thus, N2 must be

converted to more usable forms of nitrogen, such as ___________________ and ______________. The conversion of N2(g) to NH3 is called ______________________________.

2. Nitrogen fixation by prokaryotes is responsible for transforming ____________________________ of naturally occurring usable nitrogen forms. (See the diagram of the nitrogen cycle, next page.)

3. Common nitrogen-fixing bacteria found in soils include the _________________________ and the ________________-fixing bacteria. The free-living, nitrogen-fixing bacteria include

_______________________________, and cyanobacteria. a. ___________________________

i. Azotobacter is the primary species that will be selected in/on nitrogen-free media. It is a _____________________ nitrogen fixer.

ii. Only organisms that can fix N2(g) will grow on/in nitrogen-free media.

iii. Mannitol is the energy source.

iv. Molybdenum ions are included as cofactors to the nitrogenase enzyme. b. Rhizobium

i. The symbiotic nitrogen-fixing bacteria in soils include the __________________________, which form a symbiotic relationship with ______________________________________ in what is commonly referred to as _________________________________________.

Scanning electron micrograph demonstrating the colonization of wheat roots by strains of Azospirillium.

Photo courtesy of Dr. Wanjiru Mwatha

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General and Medical Microbiology Lab ■ 227 ii. The rhizobia complex includes Rhizobium, Sinorhizobium, Azorhizobium, and Bradyrhizobium,

which are common in soil and are able to fix N2

______________________________________ of legumes. Nitrogen-fixing root nodule bacteria, such as Bradyrhizobium, present inside the nodule provide valuable

____________________________________ to the host plant, which promotes plant growth. The host plant provides __________________________________________ to the bacteria.

Note: Great thanks to Dr. Wanjiru Mwatha for providing expertise for this lecture.

How Does It Work? The Nitrogen Cycle

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Experiment 22A:

Enumeration of Microorganisms in Soil

Three different kinds of media will be used to isolate 3 different types of microorganisms from soil: molds, Streptomyces, and other bacteria. Each laboratory bench will be assigned one of the three organisms to isolate. Individual groups may then choose to isolate the assigned microbe from a particular type of soil, such as barnyard soil, soil from under a tree in a park, prairie soil, etc.

Lab Period One

1. Isolation of bacteria

Procedure

From the side bench, collect one 15 mL falcon tube containing 10 mL of sterile, distilled water; three water blanks containing 99 mL of sterile, distilled water; and four TSA plates.

1. Weigh out 1 gram of soil and add it to the 15 mL falcon tube containing 10 mL of sterile, distilled water. Use the vortex to mix vigorously for 30 seconds. This ensures that bacteria are suspended in the aqueous solution and do not remain adhered to inorganic soil particles. After mixing is complete, measure the volume of the soil/water sample using the graduations on the side of the falcon tube. Record this volume in the space provided in the results section. Dilute and plate the sample as follows:

2. Label the plates 1-4, as seen in the above diagram. *If helpful label each plate with the plating factor. 3. Pipette 0.1 mL from the appropriate dilution bottle and spread this aliquot onto the correctly labeled plate with a flamed hockey stick.

4. Invert the plates and place them in the tray on the side bench to be incubated at 37°C.

2. Prescott’s Microbiology, Ninth Editon by Willey, Joanne M, Sherwood, Linda M, and Christopher J Woolverton,

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2. Isolation of Streptomyces

Streptomyces belongs to a group of Gram-positive bacteria called “actinomycetes.” They resemble fungi in appearance because they form filamentous growth. These organisms produce hundreds of different antibiotic substances; for example, Streptomyces griseus is the source of the antibiotic streptomycin. Streptomyces species also produce a compound called geosmin, which gives soil its characteristic odor. Jensen’s medium is used to enrich for Streptomyces spp.

Procedure

From the side bench, collect one 15 mL falcon tube containing 10 mL of sterile, distilled water; three water blanks containing 99 mL of sterile, distilled water; and 4 sterile, empty petri plates.

1. Weigh out 1 gram of soil and add it to the 15 mL falcon tube. Use the vortex to mix vigorously for 30 seconds. This ensures that bacteria are suspended in the aqueous solution and do not remain adhered to inorganic soil particles. After mixing is complete, measure the volume of the soil/water sample using the graduations on the side of the falcon tube. Record this volume in the space provided in the results section. Dilute and plate the sample as follows:

2. Label the 4 petri dishes 1-4, as seen in the above diagram.

3. Pipette 1.0 mL from each dilution bottle in the appropriately labeled EMPTY petri dish.

4. Pour approximately 25 mL of Jensen’s molten medium — found in the 50°C water bath (invert the container of medium three times before pouring) — onto each plate. Mix gently and allow the agar to harden.

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3. Isolation of molds

Many types of fungi can be found in soil. They are particularly important in decomposition and mycorrhizal species help plants to take up essential nutrients from the soil. Martin’s medium is formulated for the growth of molds. It contains streptomycin and rose Bengal dye to inhibit the growth of bacteria and other soil organisms. It also has a high carbohydrate content, which is favored by molds.

Procedure

From the side bench, collect one 15 mL falcon tube containing 10 mL of sterile, distilled water; three water blanks containing 99 mL of sterile, distilled water; and 4 sterile, empty petri plates.

1. Weigh out 1 gram of soil and add it to the 15 mL falcon tube. Use the vortex to mix vigorously for 30 seconds. This ensures that fungi are suspended in the aqueous solution and do not remain adhered to inorganic soil particles. After mixing is complete, measure the volume of the soil/water sample using the graduations on the side of the falcon tube. Record this volume in the space provided in the results section. Dilute and plate the sample as follows:

2. Label the 4 petri dishes 1-4, as seen in the above diagram.

3. Pipette 1.0 mL from each dilution bottle in the appropriately labeled EMPTY petri dish.

4. Pour approximately 25 mL of Martin’s molten medium — found in the 50°C water bath (invert the container of medium three times before pouring) — onto each plate. Mix gently and allow the agar to harden.

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Lab Period Two

1. Isolation of bacteria

Procedure

1. Collect the plates from the soil lab. Count the number of colonies and record these numbers in the results section of this experiment. Determine the number of organisms per gram based on your dilution series. Write this data on the board for the entire class to collect.

2. Isolation of Streptomyces

Procedure

3. Isolation of molds

Procedure

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Experiment 22A:

Results

Type of organism measured in soil: _________________________________ Type of soil used: _________________________________

Volume of the initial soil/water mixture (mL): _________________________________

1. Count the number of colonies on each dilution plate (30-300 colonies). Record the results in the table below.

Plate number Number of _colonies Titer (Number of organisms/mL of initial solution) Soil concentrations (grams of soil/mL of initial solution)* Reciprocal of the soil concentration (mL of initial solution/grams of soil) Number of organisms/gram of soil** 1 2 3 4

*Record the number of grams of soil (1 g in all cases) over the volume of the initial soil/water mixture (recorded above). This

number is the same for all plates.

**To express the number of organisms per gram of soil, multiply the titer (organisms/mL) by the reciprocal of the soil concentration (g/mL).

2. Compile the class data for enumeration of soil organisms.

Organisms Organisms/gram of soil Source: ___________________ Organisms/gram of soil Source: ___________________ Organisms/gram of soil Source: ___________________ Bacteria Streptomyces Fungi

3. Which group of organisms is present in the largest numbers?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 4. Do you think this is an accurate measurement of the number of organisms present in soil? Explain.

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

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General and Medical Microbiology Lab ■ 233 5. What are some of the sources of error (e.g. instrument error) experienced in this procedure? How could this procedure be made more volumetric?

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 6. What general types of soil microorganisms will not appear in the culture conditions used in this experiment? ______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________

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Experiment 22B:

Enrichment

of

Azotobacter

Azotobacter, a nitrogen-fixing bacteria, can be isolated by using an enrichment medium that lacks a nitrogen source.

In this lab, we will attempt to isolate Azotobacter from a soil sample by using an enrichment medium that lacks a nitrogen source. The absence of nitrogen will favor the growth of organisms that can fix atmospheric nitrogen. Mannitol is included in the medium as an energy source and an ion of molybdenum that is essential for nitrogen fixation. This metallic ion appears to serve as an essential ion cofactor for the enzyme nitrogenase, which catalyzes the reduction of nitrogen to ammonia.

Lab Period One

Procedure

From the side bench, collect 1 bottle containing nitrogen-free medium.

1. Weigh out about 1 gram of soil and place the bottle containing the nitrogen-free medium. 2. Tighten the bottle cap and shake the bottle vigorously for 30 seconds.

3. Loosen the bottle cap and place the bottle in the bin on the flat side with the neck pointed up. The medium surface should be exposed to as much air as possible as, in order to fix N2, Azotobacter requires

access to atmospheric gases.

4. Incubate the bottles at 37°C for 24-48 hours.

Lab Period Two

Procedure

From the side bench, collect 1 nitrogen-free agar plate.

1. Collect your bottle of soil culture on a nitrogen-free medium. Using an inoculating loop, collect a sample of the culture and streak for isolation onto a nitrogen-free agar plate.

Lab Period Three

Procedure

1. Collect the nitrogen-free agar plates and check for colonies. Record a description of the colony

morphology in the results section for this experiment. *Colonies will appear clear white in color and can be difficult to see

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Experiment 22B:

Results

1. Describe the colony morphology of Azotobacter.

______________________________________________________________________________________ ______________________________________________________________________________________ ______________________________________________________________________________________ 2. Draw the organisms after Gram-staining.

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Lecture 23

Tasks for the day:

Cover Lecture 23 material. Vodcast found at: https://youtu.be/rSOSKImswrs Perform Experiment 21A: Water Microbiology, Lab Period Three

Perform Experiment 22A: Enumeration of Microorganisms in Soil, Lab Period Two Perform Experiment 22B: Enrichment of Azotobacter, Lab Period Two

Perform Experiment 23A: Food Microbiology, Lab Period One Perform Experiment 23B: Caries Susceptibility Test, Lab Period One

I. Revisit to Water Microbiology

A. Multiple Tube Fermentation Method (MTFM)

Confirmed Test

MacConkey/MUG

1. Select a fluorescent colony that ferments lactose (bright pink). Use this colony to inoculate a lactosebroth tube (the ___________________________).

2. Select a second, lac+ colony and use it to inoculate the ___________________________. Choose either a fluorescent colony (presumed fecal) or a colony that does not fluoresce (presumed non-fecal).

I. Soil Microbiology

A. Isolation of differential types of soil microorganisms

1. Count the colonies on the plates used to select for growth of molds, Streptomyces species and other bacteria.

Knowledge check: Only plates containing between what two numbers of colonies should be counted? _____________________________

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General and Medical Microbiology Lab ■ 237 2. Calculate the number of organisms per gram of soil:

titer ( organisms / mL ) X

= number of organisms/g of soil

Knowledge check: Does the number recorded above represent the actual concentration of microorganisms in soil? Why or why not?

___________________________________________________________________________ ___________________________________________________________________________

B. Isolation of nitrogen-fixing bacteria

1. A nitrogen-free medium _____________________________ contain an inorganic source of nitrogen. Therefore, organisms that can live on this medium must be able to utilize

_________________________________________________. 2. Take a heavy inoculum of the nitrogen-free broth and

________________________________________________________________.

3. The major genus represented on this plate will be _____________________________________. a. Azotobacter are nitrogen fixers that are found in most

______________________________________________________________.

b. They are large, ______________________________________________ that have the ability to form resting structures called __________________________________.

II. Food microbiology

A. Food ______________________________

1. Species of _____________________________, Erwinia, and many lactic acid bacteria are common spoilage bacteria.

2. While spoilage bacteria lead to distasteful, smelly food, they are generally

______________________________________________________. However, a large number of spoilage microorganisms can indicate the presence of a

_____________________________________________________. B. Foodborne ___________________

1. ________________________ __________________________

a. Results from the ingestion of a pathogen-produced

______________________ that contaminates food.

b. Staphylococcus aureus produces an exotoxin that is an

___________________. It causes ____________________ ________________.

c. Clostridium botulinum produces an exotoxin that is a

__________________ and can cause the

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General and Medical Microbiology Lab ■ 238 2. ________________________________

a. Requires the consumption of the ______________________. Ingested bacteria will grow, invade, and often secrete toxins. This leads to _________

____________________________________________. b. Bacteria such as _____________________________, and Escherichia coli 0157:H7 cause food infections.

C. Food production: ____________________________________ 1. The starter culture of cheddar cheese is a mixture of

__________________________ spp. The starter culture of

____________________ is a mixture of Streptococcus thermophilis and Lactobacillus bulgaricus.

2. ______________________ species are also used to make sour cream and yogurt.

3. Acetobacter is used in the production of ________________________.

D. Food ___________________________ 1. Lactobacillus produces

_____________________________________, which is often used as a food preservative.

2. The lactic acid bacteria, also known as LAB, produce small antimicrobial peptides called ___________________________. These bacteriocins help the LAB compete in the food medium on which they grow. Some of the bacteriocins are being used

commercially to help preserve food.

E. Antimicrobial substances that are found naturally in food 1. Food product: ________________________

a. ____________________ targets bacterial membranes

b. Lactoperoxidase enacts _____________________________ 2 Food product: ________________________

a. ______________________________ targets peptidoglycan 3. Food product: _______________ (cloves, allspice, oregano, rosemary, sage, and vanilla)

a. _______________________ denature proteins and perturb membranes

4. Food product: ________________________

a. Thiosulfates inhibit nucleic acid and protein synthesis b. ______ is thought to inhibit bacterial metabolism. 5. Food product: ________________________

a. __________________________ are thought to inhibit DNA gyrase, damage membranes, and inhibit catabolism.