Summer Medical Microbiology Practicum 1999

 

CONTENT:

A. PROGRAM OBJECTIVES

B. OVERVIEW OF THE FIRST YEAR (1999)

C. BACTERIOLOGY

    1. Introduction
    2. Approaches to Safety in the Laboratory
    3. Preparation of Media
    4. Staining Methods
    5. Laboratory Exercise: Identification
    6. Laboratory Exercise: Sensitivity Testing

D. MOLECULAR BIOLOGY

E. OTHER AREAS COVERED

F. USEFUL REFERENCES

v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v v

A. PROGRAM OBJECTIVES:
  1. To give high school students more knowledge in medical microbiology and an opportunity to explore the career potential in this field.
  2. To provide a way for biology teachers to refresh their knowledge of microbiology and learn ways of making microbiology more meaningful and interesting to their students.
  3. To present high school students and teachers with an opportunity to join collaborations with University faculty for longer term research projects.
  4. To continue building cooperative ventures with the Des Moines Schools by sharing the scientific expertise of the University in a meaningful and useful way.
  5. To show the University’s medical students that the role of the physician encompasses teaching and research.

B. OVERVIEW OF THE FIRST YEAR (1999)

The first session was for high school students (most of the students were juniors and seniors). The session was conducted from June 14 through July 12, 1999. The second session was for high school teachers. It was conducted from July 12-21, 1999.

The program was delivered in the form of lectures, videos, and hands on. The student participants rotated through the following programs: Bacteriology, Immunology, Parasitology, Mycology, Virology, Central Sterile, Iowa Public Health department, Mercy hospital, Library, Physician Assistant program, Physical Therapy program, Family Practice and Nutrition and Wellness. We wanted the students to also be exposed not only to Microbiology but other programs at our university to help them in the knowledge of existence of other career's pathways. Teacher participants rotated through Bacteriology, Immunology, Parasitology, Mycology, Virology, Central Sterile, Iowa Public Health department, Mercy hospital, and the Library. High school students were awarded certificates of completion at the end of the program.

 

 

 

C. BACTERIOLOGY:
  1. Introduction

After being introduced to medical bacteriology and technique of isolation and identification, each participant was given clinical cases that they had to solve by identifying the causative agents, determining the treatment of choice by performing the sensitivity tests using the disk diffusion method, and preparing a presentation using power point. In the laboratory, students worked closely with faculty, staff and student assistants. High school students were given four cases each. Participants were not allowed to work unsupervised.

Lectures

Lectures were divided into systems with emphasis on the important infectious agents, gram reaction, habitat, mode of transmission, specimen required, laboratory diagnosis, treatment of choice and prevention. Selected agents from each system were used as unknowns in the clinical cases .

  1. Approaches to Safety in the Laboratory
    1. Orientation to the Microbiology Laboratory Instructions (Josephine A Morello et. al.,)
    2. Students watched the Universal Precautions: Aids and Hepatitis B Prevention for Home Health Care video by Medcom, Inc (1997), Toll free (800) 877-1443 Fax (714) 898-4852 . Web: http://www.medcominc.com.. This video contains CDC's 1996 "Standard Precautions" and "Transmission-Based Precautions". Students answered questions and discussed the importance of safety in thelaboratory.
    3. We used known non-pathogenic organisms. For example, to show students acid fast staining method, we used Mycobacterium segmatis. Slides were made and heat fixed before being used by students.
    4. Another strategy was to pre inoculated the biochemicals and the students just read them.
    5. We also used organisms isolated from the students' own flora (hair, mouth etc.)

References:

Fleming, D.O. et al 1995. Laboratory Safety, Principles and Practices, ASM Press, Washington, D.C.

Morello, Josephine A et. al. Laboratory Manual and Workbook in Microbiology Applications to Patient Care 5th Edition, 1994, Wm C. Brown Communications, Inc.)

3. Preparation of media:

We followed the formula from the Difco Manual (11th Edition), BD Biosciences . The Difco Manual explains the principal and use of each medium.

http://www.bdms.com//difco/manual.html

Mueller Hinton Agar

Purpose: A medium used for testing the susceptibility of microorganisms to antimicrobial agents.

EMB (eosin methylene bleu) agar

Purpose: A differential medium recommended for the detection and isolation of the gram-negative enteric bacteria.

MacConkey Agar

Purpose: A differential medium recommended for use in the isolation and differentiation of lactose-fermenting organisms from lactose nonfermenting gram-negative enteric bacteria.

Tryptic soy agar

Purpose: A general purpose medium used with or without blood or other enrichment for isolating and cultivating a variety of fastidious microorganisms.

Mannitol salt agar

Purpose: A selective medium used for the isolation of pathogenic staphylococci . Growth of Gram negative organisms and streptococci is inhibited. Staphylococcus aureus will form good to excellent yellow colonies. The other staphylococci will form poor to fair red colonies.

Blood agar

Purpose: A general purpose medium used for isolating and cultivating microorganisms (Supplier: Remel).

Chocolate agar

Purpose: An enriched medium used for isolating and cultivating fastidious microorganisms (Remel).

Lowenstein-Jensen agar

Purpose: An egg-base medium used for the primary isolation of mycobacteria (Remel.

Thioglycollate broth

Purpose: A medium used for isolation of anaerobic, microaerophilic and aerobic microorganisms. This medium used for isolation of actinomyces species. (Remel)

Yeast extract agar

Purpose: An enriched medium used for growth of nocardia. (Remel)

References:

1. Difco Manual (11th Edition), BD Biosciences . Difco Laboratories. Detroit Michigan 48232

http://www.bdms.com//difco/manual.html

2. Remel

12076 Santa Fe Dr., Lenexa, KS 66215

Tel: 800/255-6730 Fax: 913/888-5884

3. Source Book (the authority on where to find reagents, equipment, supplies and services). 2000. Published with Cold Spring Harbor Laboratory Press. BioSupplyNet. http://www.biosupplynet.com

 

 

 

4. Staining Methods:

 

Gram Stain

Principle and Procedure

Bacteria can be divided into two groups according to the difference in the morphology of the cell wall. This difference was first observed in 1884, by a Danish physician named Hans Christian Gram. He developed a new method to stain bacteria so they can be visible in specimen samples. He used the following steps, each followed by a wash:

1. A smear of the specimen (or from a culture sample) is made on a slide. The air dried slide is heat fixed.

2. The smear is flooded with crystal violet (the primary dye). All the bacteria on the smear will stain purple.

3. Iodine is added as a mordant. Iodine fixes the primary dye to the bacterial cells. All the cells will look purple.

4. Alcohol is added to decolorize the cells. One group of bacterial cells will retain the primary dye and stain purple (these are called Gram-positive). The other group won't retain the dye, it will look colorless.

5. Safranin is added to the slide. The purple stained bacteria will remain the same. The colorless bacteria will stain red (Gram-negative).

Importance of Gram Stain

This staining method is still valuable today. It is used in bacterial identification and taxonomy. It is of great importance in diagnosis of infectious diseases and in guiding drug therapy. For instance, the majority of Gram-positive organisms are susceptible to penicillin, while gram-negative bacteria are resistant to this antibiotic.

Examples:
    1. Gram stain of bacillus subtilis

      2. http://www.asmusa.org/edusrc/library/FactSheet.asp?SubmissionID=369

    2. Gram stain of Staphylococcus aureus

      3. http://www.asmusa.org/edusrc/library/FactSheet.asp?SubmissionID=387

    3. Gram stain of Neisseria gonorrhoeae

http://www.asmusa.org/edusrc/library/FactSheet.asp?SubmissionID=386

India Ink Stain

Principle

This stain is used for direct microscopic examination of capsules of microorganisms. The India ink gives a semiopaque background against which the clear capsules can be easily visualized.

Procedure
    1. Add a small loop full of sterile water on a pre-cleaned microscope slide.
    2. Place slide on staining rack.
    3. Add a small amount of bacterial growth.
    4. Add one drop of India ink and mix.
    5. Cover with a thin, large coverslip. Press gently to allow the fluid to spread as a thin film beneath the coverglass.
    6. Examine with high-dry and oil immersion lens.

Interpretation

Capsules appear as clear zones (halos) around the refractile organism.

Examples:

Bacteria with capsules: Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas putida.

Image: http://www.asmusa.org/edusrc/library/images/jbowen/Images/jbowene2.jpg

References:

    1. Brochure from Becton Dickinson Microbiology Systems, Cockeysville, Maryland.
    2. Koneman, E.W., et al. 1997. Color Atlas and Textbook of Diagnostic Microbiology. (5th ed.) J.P. page 904, Lippincott Co., Philadelphia.

Kinyoun Stain (TB Stain Kit K)

Principle

Mycobacteria species contain mycolic acid (wax) in their cell wall. They are "acid-fast". It means once stained with an aniline dye such as carbolfuchsin, they are difficult to decolorize, and retain the red color even when treated with a mixture of acid and alcohol.

Procedure
    1. Cover an air dried, heat-fixed smear with a small rectangle of filter paper.
    2. Place slides on a staining rack and flood with TB Carbolfuchsin KF for 4 min. Do not heat.
    3. Remove filter paper with forceps, wash gently in running water.
    4. Decolorize with TB Decolorizer for 3-5 sec (until nor more red color rinses off the slide).
    5. Wash gently in running water.
    6. Counterstain with TB Brilliant Green K for 30 sec.
    7. Wash gently in running water.
    8. Air dry.
    9. Examine with oil immersion lens.

Interpretation

Acid-fast Bacilli = Dark red to pink (Example: Mycobacterium smegmatis, this is a non-pathogenic organism)

Non-acid-fast Bacilli = green (Example: Escherichia .coli, Staphylococcus)

Reference
    1. Brochure from Becton Dickinson Microbiology Systems, Cockeysville, Maryland
    2. Koneman, E.W., et al. 1997. Color Atlas and Textbook of Diagnostic Microbiology. (5th ed.) J.P. page 904, Lippincott Co., Philadelphia.

 

 

 

 

Partial Acid-Fast Stain for Identification of Nocardia

 

Principle

The nocardiae, because of mycolic acid in their cell walls, can retain carbolfuchsin dye during mild acid decolorization, whereas other branching bacilli cannot.

Procedure
    1. Emulsify a very small amount of organisms in a drop of distilled water on the slide.
    2. Place slide on the staining rack.
    3. Allow to air dry and heat fix.
    4. Place a small filter paper on top of the smear
    5. Flood the stain with Kinyoun’s carbolfuchsin for 3 minutes.
    6. Remove the small filter paper
    7. Wash gently in running water.
    8. Decolorize briefly with 1% sulfuric acid until no more red color rinses off the slide.
    9. Counterstain with Kinyoun’s Brilliant Green K for 30 sec.
    10. Wash gently in running water.
    11. Air dry.

Interpretation

Acid-Fast Bacilli Nonacid-Fast Bacilli

Reddish to purple filaments green

Reference:

Baron, J.E., Finegold, S.M. 1998. Bailey & Scott's Diagnostic Microbiology (10th ed). The C.V. Mosby Company, Baltimore.

Spore stain

Principle

Endospores are small and metabolically dormant structures. They contain a large amount of a chemical found only in bacteria, calcium dipicolinate, a small amount of water, DNA and tough coats surrounding the endospore. Endospores are the most resistant form of life. They present a big problem in preservation of food and in the sterilization of medical instruments. They are resistant to boiling and many chemicals. Two genera of bacteria, Bacillus and Clostridium form endospores during periods of nutrient depletion. These bacteria are found in the soil and they are of medical importance: Examples: Bacillus anthracis, agent of cutaneous or pulmonary anthrax. Wool-sorters contract this disease by inhaling spores from contaminated hair or wool of animals such as goats and sheep. Bacillus cereus, agent of food poisoning; Clostridium tetani, agent of tetanus or lockjaw; Clostridium perfringens, agent of gas gangrene; Clostridium botulinum, agent of food poisoning called botulism.

Image: http://www.microbelibrary.org/Fact Sheet.asp?SubmissionID=582

 

Schaeffer-Fulton Staining Method [(0.5% (wt/vol) aqueous malachite green]

Procedure:

    1. Make a smear on a glass slide.
    2. Place slide on staining rack.
    3. Air-dry the smear.
    4. Heat fix.
    5. Cover with a small piece of paper towel saturated with malachite green.
    6. Place over a boiling-water bath for 5 min. (do not let the smear dry, keep adding dye).
    7. Allow the slide to cool slightly.
    8. Gently wash the slide in tap water.
    9. Counterstain with safranin for 30 seconds.
    10. Wash in tap water.
    11. Blot dry.
    12. Examine under the microscope (use oil immersion).

Interpretation:

Spores = bright green (Organisms: Bacillus species)

Vegetative cells = brownish red (Organisms: Escherichia coli, Staphylococci)

Reference:

Philipp Gerhardt et., al. Methods for General and Molecular Bacteriology. Page 34. ASM Press 1994.

 
  1. Laboratory Exercise: Identification of Pyogenic Cocci and some members of the Enterobacteriaceae

 

INTRODUCTION

Pyogenic bacteria (pus-producers) and nearly all members of the family Enterobacteriaceae cause infections in virtually any tissue, organ, tract or system of the body. The clinical laboratory must be prepared to identify these organisms rapidly and accurately because of their clinical and epidemiological importance, as well their differing antibiotic susceptibility patterns.

Included among the pyogens are members of the genera Enterococcus, Neisseria, Staphylococcus, and Streptococcus. The following genera: Escherichia, Shigella, Salmonella, Proteus, Klebsiella, and Enterobacter are among the members of the family Enterobacteriaceae

 

PURPOSE

The overall goals of this laboratory exercise are:

1. to acquaint the student with the Gram-stain technique and to understand its value in the study of bacterial morphology

2. to familiarize the student with some rapid methods for the identification of pyogenic cocci, and Enterobacteriaceae.

OBJECTIVES

At the end of this laboratory, the student should:

1. Know how to perform and interpret the Gram-stain

2. Know the principle and interpretation of tests used.
    1. Be able to biochemically identify the organisms used.

Media for isolation

Central Nervous system specimen: blood agar (BA), eosin methylene blue (EMB) and

chocolate

Genital tract specimen: BA, EMB and chocolate

Respiratory tract specimen: BA, EMB and chocolate

Stool specimen: EMB, MacConkey, BA

Urinary tract specimen: BA, EMB

Identification

We used conventional biochemical tests as well as rapid pre-packaged kits.

PROCEDURE

Day 1
    1. Perform a Gram-stain on your isolate and record the result.
    2. Streak your gram positive organisms and gram negative rods on Tryptic Soy Agar slants. Incubate at 370C. Streak your gram-negative cocci on chocolate agar. Incubate in CO2 incubator.
    3. Record the colonial morphology of the isolate.
    4. Plate the isolates on media according to the source of the specimen.
    5. Incubate in appropriate incubator.

Day 2
    1. Put your inoculated slants and chocolate agar in the refrigerator to preserve your unknowns.
    2. Use the appropriate tests to identify your isolates. The following Flow Charts (I-IV) and Table 1 will guide you in the identification of the unknown.
    3. Incubate the isolates in an appropriate incubator.
    4. Identify your gram positive rods at the GENUS LEVEL only by doing the spore stain.

Day 3
    1. Read your results and identify your isolate to the species level.
    2. The principle of each test, performance and interpretation are provided.

 

FLOW CHART I

SCHEMATIC GUIDE TO THE IDENTIFICATION OF THE STAPHYLOCOCCI

 

Gram positive cocci

CATALASE +

Staphylococcus

 

COAGULASE + COAGULASE -

S. aureus

(others)

 

Novobiocin Novobiocin

sensitive resistant

S. epidermidis S. saprophyticus

(others) (others)

Image of Staphylococcus aureus

http://www.asmusa.org/edusrc/library/images/tomalty/HTMLpages/STAPHAUR.HTM

 

 

 

 

 

 

 

 

 

 

FLOW CHART II

SCHEMATIC GUIDE TO THE IDENTIFICATION OF STREPTOCOCCI

 

Gram positive cocci

CATALASE -

Streptococcus

 

TYPE OF HEMOLYSIS (http://www.asmusa.org/edusrc/library/images/tomalty/Images/beta.jpg)

ALPHA BETA

1) Optochin sensitive 1) Bacitracin sensitive

S. pneumoniae S. pyogenes

 

2) CAMP +

S. agalactiae

GAMMA

 

BILE ESCULIN +, SALT BROTH + BILE ESCULIN +, SALT BROTH -

E. faecalis S. bovis

 

Image of Enterococcus (http://www.asmusa.org/edusrc/library/images/tomalty/HTMLpages/ENTERO.HTM)

Image of Streptococcus pneumoniae (http://www.asmusa.org/edusrc/library/images/tomalty/Images/Spneumo.jpg)

Image of Streptococcus pyogenes (group A strep) pharyngitis

http://www.asmusa.org/edusrc/library/images/tomalty/HTMLpages/STREP.HTM

*Confirmation of group A Strep, perform the STREP A TEST.

 

CHART III: SCHEMATIC GUIDE TO THE IDENTIFICATION OF GRAM NEGATIVE COCCI

Gram negative Cocci or Coccobacilli

Oxidase +

Neisseria or Moraxella

Sugar fermentation

Organisms

Glucose

maltose

sucrose

lactose

Neisseria gonorrhoeae

+

-

-

-

Neisseria meningitidis

+

+

-

-

Moraxella catarrhalis

-

-

-

-

Unknown

        

Image of Neisseria gonorrhoeae http://www.asmusa.org/edusrc/library/images/tomalty/Images/NEISSER-an.jpg

Commercial identification kit: API QuadFERM + system (BioMérieux Vitek, 595 Anglum Rd. Hazelwood, MO 63042-2320

Tel: 314-731-8500, 800-638-4835, Fax: 800-325-1598

http://194.250.199.133/english/bioweb-eng.html

FLOW CHART IV: SCHEMATIC GUIDE TO IDENTIFICATION OF ENTEROBACTERIACEAE

Gram negative rods

OXIDASE -

API 20E system (Rapid identification kit from BioMérieux )
 

ONPG ADH LDC

1 2 4

ODC CIT H2S

1 2 4

URE TDA IND

1 2 4

VP GEL GLU

1 2 4

MAN INO SOR

1 2 4

RHA SAC MEL

1 2 4

AMY ARA OXI

1 2 4

Example

+ + +

- + -

- - +

+ + +

+ - -

- + -

+ - +

Unknown

              

      

Profile number (example) 7247125 ID: Aeromonas hydrophila

      

Profile number (Unknown) ------- ID:

Table 1: SUMMARY OF THE API-20E SYSTEM RESULTS

 

Code

Test

Positive Reaction

Negative Reaction

ONPG

Hydrolysis of o-nitrophenyl-B-galactopyranoside

Yellow

Colorless

ADH

Arginine dihydrolase

Red or Orange

Yellow

LDC

Lysine decarboxylase

Red or Orange

Yellow

ODC

Ornithine decarboxylase

Red or Orange

Yellow

CIT

Citrate utilization

Turquoise or

Dark Blue

Light Green

or Yellow

H2O

Production of hydrogen sulfide

Black Deposit

No Black Deposit

URE

Urea hydrolysis

Red or Orange

Yellow

TDA

Tryptophan deaminase

Brown-Red

Yellow or Orange

IND

Formation of indole

Red Ring

Yellow

VP

Production of acetoin

(Voges-Proskauer test)

Red

Colorless

GEL

Liquefaction of gelatin

Pigment Diffusion

No diffusion

GLU

MAN

INO

SOR

RHA

SAC

MEL

AMY

ARA

Fermentation of:

Glucose

Mannitol

Inositol

Sorbitol

Rhamnose

Sucrose

Melibiose

Amygdalin

Arabinose

Yellow or Gray

Blue or Blue-Green

OXI

Presence of cytochrome

Oxidase

Blue-Purple

No Blue-Purple

 

 

Principles of Tests Used

BACITRACIN INHIBITION TEST

Principle

The purpose of the test is to differentiate group A beta hemolytic streptococci from other groups. A bacitracin disk containing 0.04 units will inhibit group A streptococci while most other beta hemolytic streptococci are not inhibited. The test is designed for use on a pure culture and not for direct use on the primary isolation plate.

Performance of the test
    1. Streak beta hemolytic Streptococcus heavily on area of a sheep blood agar plate. Place bacitracin disk in center of the streak and tap down to ensure surface contact.
    2. Incubate for 18-24 hrs at 350C in a non CO2 incubator. Read for inhibition.

Interpretation: Any zone of inhibition is read as a positive test.

CAMP TEST

Principle

The hemolytic activity of staphylococcal B-lysin on erythrocytes is enhanced by an extracellular factor produced by group B streptococci, called the CAMP FACTOR (acronym for Christie, Atkins, and Munch-Peterson who reported this phenomenon in 1944). The blood agar plate contains one streak of B-lysin producing Staphylococcus aureus and two streaks of the isolate.

Interpretation:

Positive: Presence of an arrow zone of hemolysis at the point where the perpendicular streaks meet.

Negative: No arrow.

BILE ESCULIN TEST

Principle

This test is based on the ability of some bacteria, especially group D streptococci, to hydrolyze esculin in the presence of 1% to 4% bile salts. Esculin, a glycoside, is hydrolyzed by bacteria into esculetin and glucose. Esculetin reacts with an iron salt to form a dark brown or black complex, resulting in a diffuse blackening of the bile-esculin medium, which contains ferric citrate as the source of ferric ions.

Interpretation

Positive: Production of black colored compound.

Negative: No production of black colored compound.

CATALASE TEST

Principle

This test is used to detect the presence of the enzyme CATALASE which splits hydrogen peroxide into water and oxygen

catalase

2H2O2 --------> 2H2O + O2

Specimen

Culture of bacteria growing on blood agar plate.

Procedure

  1. Using a wooden stick, transfer a small amount of growth from surface of colony on blood agar (avoid carry over of medium) onto the surface of a clean, dry glass slide.

  2. Place a drop of 3% hydrogen peroxide (H2O2) on the bacterial smear.

Result and Interpretation

POSITIVE = Immediate and vigorous bubbling due to conversion of H2O2 to water and oxygen.

NEGATIVE = no bubbling

 

COAGULASE TEST

Principle

Coagulase is a protein with a prothrombin-like activity. This protein can convert fibrinogen into fibrin resulting in a clot formation.

Specimen

Culture of bacteria growing on blood agar plate.

Procedure

a) Using a loop transfer a small amount of growth from a well isolated colony into a tube of rabbit plasma.

b) Gently rub the growth against the side of the tube as to form a uniform suspension of the organisms in the plasma.

c) Incubate at 37oC for 4 hrs -24 hrs. Use a non CO2 incubator.

Result and Interpretation

POSITIVE = Clot formation (any degree of clot formation is considered positive)

NEGATIVE = no clot

NOVOBIOCIN SCREENING TEST

Principle

This test is used to screen coagulase negative staphylococci from urine specimens to detect S. saprophyticus. Of the clinically significant coagulase negative staphylococci, S. saprophyticus is resistant to 5 ug/of Novobiocin.

Procedure

    1. Streak staphylococci colonies heavily on area of a sheep blood agar plate using a cotton swab. Place novobiocin disk in center of the streak and tap down to ensure surface contact.
    2. Incubate for 18-24 hrs at 350C in a non CO2 incubator. Read for inhibition.

Interpretation

Sensitive = Diameter zone of inhibition >/16mm

Resistant = Zone of inhibition < 16mm

 

 

 

 

 

OPTOCHIN SUSCEPTIBILITY TEST

Principle

Ethylhydroxycupreine hydrochloride (Optochin) is a quinine derivative which selectively inhibits the growth of Streptococcus pneumoniae in very low concentrations (5 mcg/ml or less). Optochin can inhibit other alpha-hemolytic streptococci, only at higher concentrations.

Interpretation

POSITIVE (Sensitive) = Diameter zone of inhibition of 16mm or greater

NEGATIVE (Resistant) = Diameter zone of inhibition less than 16mm

OXIDASE TEST

Principle

This is a test for the presence of cytochrome oxidase, an iron-containing enzyme which participate in the electron transport system and the nitrate metabolic pathways.

Specimen

Culture of bacteria growing on blood agar plate.

Procedure

a) Wet filter paper with a drop of oxidase reagent.

b) With a wooden applicator pick a colony of the isolate and rub on the oxidase wetted paper.

Result and Interpretation

POSITIVE = Development of a blue-purple color within 30 seconds.

NEGATIVE = No blue-purple color.

SALT TOLERANCE TEST

Principle

Certain bacteria, especially the Group D enterococci tolerate and are able to grow in the presence of 6.5% sodium chloride.

Procedure

  1. With a loop, pick 2 or 3 colonies of organisms growing on blood agar

  2. Inoculate a salt broth

  3. Incubate at 370C for 24 hours

  4. Read broth

Interpretation

Positive = Turbidity in the tube indicates growth

Negative = No turbidity

 

 

 

 

 

 

PACKAGED RAPID MICROBIAL IDENTIFICATION SYSTEMS

QUADFERM+

Principle:

The quadFerm+ system is intended for the rapid determination of carbohydrate utilization, DNASE activity and penicillinase activity by Neisseria and Moraxella species.

The quadFerm+ system consists of seven test microcupules, which are rehydrated with a bacterial suspension in 0.85% saline. This system is an acidimetric method for detection of carbohydrate utilization and DNase and penicillinase activity using phenol red as the pH indicator. Utilization of the substrate produce a color change in the phenol red indicator because of the production of an acid environment.

Reference

API QuadFERM + system (BioMérieux Vitek, 595 Anglum Rd. Hazelwood, MO 63042-2320

Tel: 314-731-8500, 800-638-4835, Fax: 800-325-1598

http://194.250.199.133/english/bioweb-eng.html

 

API 20E SYSTEM

Principle

The API 20E system is a standardized miniature version of conventional biochemical procedures for the identification of Enterobacteriaceae and other Gram negative bacteria. The system consists of microtubes containing dehydrated substrates. The substrates are reconstituted when a bacterial suspension is added. It is incubated so that the organisms react with the tubule contents and are read when various indicator systems are affected by the organisms metabolites or added reagents after 18-24 hrs.

Reference

API QuadFERM + system (BioMérieux Vitek, 595 Anglum Rd. Hazelwood, MO 63042-2320

Tel: 314-731-8500, 800-638-4835, Fax: 800-325-1598

http://194.250.199.133/english/bioweb-eng.html

 

STREP A TEST

Principle

Strep A test is used for the qualitative detection of Group A streptococcal antigen from throat swabs or confirmation of Group A streptococcus for colonies recovered from blood agar. The ACCURACY.EASE.VALUE (Acceava) Strep A test can detect viable or nonviable organisms directly from a throat swab. The result are ready within 5 minutes. The ACCEAVA test uses a color immunochromatographic dipstick containing rabbit antibodies coated on a nitrocellulose membrane. Group A streptococcus in the sample will form a complex with the anti-Group A streptococcus antibody conjugated color particles. The complex will be bound by the anti-Group A streptococcus capture antibody. A positive reaction is indicated by a blue test line.

Reference: BIOSTAR, Manufactured for BioStar. Inc, Boulder, Colorado USA

Telephone : 303.530.3888 Toll Free: 800.637.3717 Fax: 303.530.6601

URL: http://www.biostar.com/

6. Sensitivity Testing:

The mechanism of action and spectrum were explained. Participants made Mueller Hinton agar for sensitivity testing. The principal and use of the E-test were shown to students. They performed the disk diffusion test on their unknown using the following antibiotics: ampicillin, ticarcillin, cefazolin, gentamicin, sulfamethoxazole-trimethoprim, nitrofurantoin, penicillin, vancomycin and erythromycin.

 

Procedure for Performing the Disk Diffusion Test

I. Direct Colony Suspension Method
    1. Transfer an isolated bacterial colony from a blood agar plate to a tube containing broth or saline to make a bacterial suspension. Mix tube well to disperse the colony.
    2. Check the turbidity of the suspension matches the provided 0.5 McFarland turbidity standard. If the suspension is less turbid than the standard, transfer additional colonies until the suspension matches the standard.

II. Inoculation of Test Plates

  2. 1. Transfer a portion of the adjusted suspension to a Mueller Hinton agar plate, using a sterile cotton swab. Dip the swab into the suspension, rotate the swab several times and press the swab against the inside wall of the tube above the fluid level to remove extra inoculum.
  3. 2. Streak the swab over the entire surface of the agar, then repeat twice, rotating the plate at 60° each time to evenly spread the inoculum over the agar surface.
  4. 3. Partially cover the agar plate with the plate lid and leave plate set for 3 to 5 minutes maximum while excess moisture is absorbed into the agar.

    5. III. Application of Disks to Inoculated Agar Plates

    1. When the plates have dried, dispense the antimicrobial disks to the plates using the dispensing apparatus. Distribute the disks evenly so that the centers of the disks are no closer than 24 mm apart. Do not move the disks once they have been placed on the agar, as the drugs start to diffuse as soon as they contact the agar.

    2. Invert the plates within 15 minutes after the disks have been applied, and incubate them at 35°C overnight.

    IV. Reading Plates and Interpreting Results
  5. 1. Examine incubated plates for presence and size of zones of incubation around lawns of bacterial growth.

    6. 2. Measure the diameters of the zones of complete inhibition using a sliding caliper or ruler held against the back of the plate. Round measurement to the nearest whole millimeter.

  6. 3. The edge of the inhibition zone should be the border which shows no visible bacterial growth.

Antimicrobial Gradient Strip Method (E-Test; PDM Epsilometer)

    1. Subculture bacteria to be tested by diluting colonies and streaking the diluted suspension on Mueller-Hinton plates as is done for the "Disk Diffusion" test (follow protocol steps listed in sections I. and II. of the Disk Diffusion test)
    2. Place the gradient strip antibiotic side down on the surface of the bacteria-covered agar plate. Incubate plates as is done for the Disk Diffusion test.
    1. Check the growth pattern of the bacteria. The elliptical zone of inhibition is read where it intersects the growth scale printed on the gradient strip.

Reference

1. AB Biodisk, Piscataway, N.J.

2. Remel

12076 Santa Fe Dr., Lenexa, KS 66215

Tel: 800/255-6730 Fax: 913/888-5884

MOLECULAR BIOLOGY (Dr. Travis Knight)

Overview of activities
    1. PCR differentiation of Yersinia enterocolitica isolates

      2. -streak plates

      -isolate genomic DNA

      -quantitate and check purity

      -PCR

      -analyze PCR products

    2. Isolate and identification of plasmid DNA

      3. -start overnight liquid cultures

      -isolate plasmid DNA

      -cut with restriction enzyme

      -analyze restriction fragments

    3. DNA spooling from liver, kiwi, and onion

 

Yersinia enterocolitica Data Sheet

 

Sample Number ________

 

Dilution for Spectrophotometer ________

 

260 nm Absorbance ________

 

280 nm Absorbance ________

 

260/280 Ratio ________

 

Concentration of diluted DNA ________ ug/ml

 

Concentration of stock DNA _________ ug/ml

 

Dilution needed to get to 10 ng/ul ________

 

Small-Scale Preparations of Plasmid DNA – Alkaline Lysis Minipreps

Harvest
    1. Transfer a single bacterial colony into 2 mL of LB medium containing the appropriate antibiotic in a loosely capped 15-mL tube. Incubate the culture overnight at 37° C with vigorous shaking.
    2. Pour 1.5 mL of the culture into a microfuge tube. Centrifuge at 12,000g for 30 seconds at 4° C in a microfuge. Store the remainder of the culture at 4° C.
    3. Remove the medium by aspiration, leaving the bacterial pellet as dry as possible.

Lyse
    1. Resuspend the pellet in 100 uL of ice-cold Solution I (50 mM glucose, 25 mM Tris-Cl {pH 8.0}, 10 mM EDTA {pH 8.0} autoclave and store at 4° C) by vigorous vortexing.
    2. Add 200 uL of freshly prepared Solution II (0.2 N NaOH, 1% SDS). Close tube and mix by inverting the tube rapidly five times. Make sure that the entire surface of the tube comes in contact with Solution II. DO NOT vortex.
    3. Add 150 uL of ice-cold Solution III (3 M potassium, 5 M acetate). Close the tube and flick the tube with your finger in an inverted position for 10 seconds to disperse solution III through the viscous bacterial lysate. Store on ice for 5 minutes.
    4. Centrifuge at 12,000g for 5 minutes at 4° C in a microfuge. Transfer the supernatant to a clean tube.
    5. Precipitate the DNA with 2 volumes of ethanol at room temperature. Mix by vortexing. Allow to stand 2 minutes at room temperature.
    6. Centrifuge at 12,000g for 5 minutes at 4° C in a microfuge.
    7. Remove the supernatant by gentle aspiration. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Remove any drops of fluid adhering to the walls of the tube.
    8. Rinse the pellet with 1 mL of 75% ethanol at 4° C. Repeat steps 6 and 7. Allow the pellet to dry for 10 minutes.
    9. Redisolve the nucleic acids in 20 uL TE.

Digest
    1. To a clean tube add 2 uL isolated plasmid, 1 uL RNAse, 1.5 uL 10X restriction digest buffer, 10.5 uL water, and 1 uL Taq I restriction enzyme. Gently mix and give the tube a "quick spin" to collect the liquid at the bottom.
    2. Allow the digestion to proceed for about 1 hour at 37° C.
    3. Add 5 uL blue juice and load on gel for analysis.

Reference:

Sambrook, J., Fritsch, E.F., and Maniatis, T.E. 1989. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

 

 

OTHER AREAS COVERED:

We also wanted the participants to explore other health related career opportunities offered at our University.

  • Centrale sterile services

Prevention is the key for control of infectious agents. Participants were exposed to importance of prevention through different methods of sterilization. Participants also needed to know career opportunities in this area.

Topics discussed:

    1. Personal protection
    2. Care and cleaning of surgical instruments and equipment
    3. Chemicals and MSDS
    4. Sterilization, disinfection and sanitation
    5. Methods of sterilization
    6. Monitoring of sterilization cycle
    7. What to sterilize and when
    8. Inventory (Distribution and control)
    9. Hazardous waste (Disposal and what constitutes hazardous waste)
    10. Medical Records (Confidentiality, distribution of information etc.)
  • Library Services: Participant were introduced to medline search, internet.

Website references dealing with microorganisms, books and names of journals were provided

  • Tour of Mercy Hospital

To appreciate the importance of Microbiology and clinical diagnosis, the participants made a tour of the Mercy Hospital Medical Center. Mercy Hospital is a tertiary care hospital that has a family practice residency program affiliated with Mayo Hospital. The hospital has 636 beds and a high complexity laboratory. The participant toured the following laboratory sections: chemistry, microbiology, hematology, blood bank, cytology, and immunology. Ms. Stacy Sime, the Interim Program Director of Mercy School of Clinical Laboratory Science and Technology conducted the tour.

 

 

 
  • Iowa Public Health Department Visit:

The participants visited the Iowa Public Health Department. Dr. Patricia Quinlisk, Iowa's epidemiologist, and her residents discussed with the participants the role of the Public Health Department, recent outbreaks in Iowa and the importance of prevention.
  • Family Practice Department: students made rounds with 3rd year osteopathic students.
  • Physician Assistant (PA) program: Student learned what a PA is, how the education is UOMHS, hands on training of typical PA duties, simple suture techniques, physical exam techniques and use of "Harvey" the heart sounds simulator.

USEFUL REFERENCES:

Websites:

Johns Hopkins University School of Medicine – case rounds from the weekly infectious Disease Case Conference.

http://hopkins-id.edu/education/id_caserounds/index_case.html

Infectious Disease WebLink (I.D.W.L), clinical images in infectious disease

http://pages.prodigy.net/pdeziel/case.htm

Excellence in Curriculum Integration Through Teaching Epidemiology (EXCITE)

http://www.cdc.gov/excite/

http://www.cdc.gov/excite/iowa.htm

CDC Morbidity and Mortality Weekly Report

http://www2.cdc.gov/mmwr/

CDC Facts Sheets from MMWR

http://www.cdc.gov/od/oc/media/facts.htm

Hygienic Laboratory, The University of Iowa

http://www.uhl.uiowa.edu/

http://resistanceweb.mfhs.edu/cit/about_resistance_web/about_resistance_web.asp

National Necrotizing Fasciitis Foundation

http://www.nnff.org./

http://www.cnn.com/HEALTH

http://www.emedicine.com/emerg

http://www.hosppract.com

 

Books

Fauci, A.S., et.al., (eds), Harrison's Principles of Internal Medicine, 14th edition, McGraw-Hill, 1998.

Goldman, L. and Bennett, J.C. (eds.), Cecil's Textbook of Medicine, 21st edition, W.B. Saunders, 2000.

Chin, James, editor: Control of communicable diseases manual : an official report of the American Public Health Association . Edition 17th ed. Imprint Washington, DC : American Public Health Association, 2000.

Murray, P.R., et al 1998, Medical Microbiology , 3rd edition Mosby Yearbook, Inc. St. Louis MO

Gilligan, PH., et al 1997 Cases in Medical Microbiology and Infectious Diseases (2nd edition), ASM Press, Washington, D.C.

Bartlett, J.G. 1998, Pocket Book of Infectious Disease Therapy, Williams & Wilkins, A Waverly Company, Baltimore.

Fleming, D.O. et al 1995. Laboratory Safety, Principles and Practices, ASM Press, Washington, D.C.

Koneman, E.W., et al. 1997. Color Atlas and Textbook of Diagnostic Microbiology. (5th ed.) J.P. Lippincott Co., Philadelphia.

Baron, J.E., Finegold, S.M. 1998. Bailey & Scott's Diagnostic Microbiology (10th ed). The C.V. Mosby Company, Baltimore.

Difco Manual (10th Edition), 1984. Difco Laboratories. Detroit Michigan 48232

Source Book (the authority on where to find reagents, equipment, supplies and services). 1999. Published with Cold Spring Harbor Laboratory Press. BioSupplyNet on the web at www.biosupplynet.com

Other:

National Laboratory Training Network Midwestern Office

2121 W. Taylor Street, Chicago, IL 60612

Phone: 312/793-3306 or Fax: 312/793-3304

Journal

The New England Journal of Medicine

Emerging Infectious Diseases (Department of Health and Human Services, CDC)

Faculty and Staff

Coordinator

Musau WaKabongo, Ph.D.

Associate Professor of Microbiology

(515) 271-1621

Fax: (515) 271-7035

E-mail: Musau.Wakabongo@dsmu.edu

http://www.dsmu.edu/microbiology/index.htm

Des Moines University Faculty and Staff

Ellen Bergerson, Assistant Librarian

Susan Bravard, P.T., Assistant Professor of Physical Therapy

Lola Deuel, Central Sterile Processing Technician/Supervisor

James Johnson, Ph.D., Professor of Microbiology

Travis Knight, Ph.D. Postdoctoral Fellow, Research

Gregory Kolbinger, P.A.-C, Assistant Professor, Physician Assistant Program

Bryan Larsen, Ph.D., Professor of Microbiology, Dean of Research

Tom Mueller, Ph.D., Professor of Biochemistry, Chair, Microbiology Department

Deborah Potym Torbert, BS, MT (ASCP), Medical Technologist, Microbiology

Juanita Robel, M.H.S., P.T., Assistant Professor of Physical Therapy

Joy Schiller, M.S. Adjunct Assistant Professor, Health Care Administration

Karen Smith, Technical Services Librarian

Fred Strickland, M.A., D.O., Professor of Family Practice

DMU Student Participants

Tuan Ward, M.S., D.P.M. (DO 2002)

Amy Frey, M.S. (DO 2002)

Guest Participants

Patricia Quinlisk, M.D., M.P.H., Iowa State Epidemiologist

Stacy Sime, MS, MT (ASCP), SBB, Interim Program Director,

Mercy School of Clinical Laboratory Science and Technology