Lab Manual 2012-13

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Medical School for International Health

CLINICALBACTERIOLOGY

LABORATORYfor

Microbiology 2012-2013

LABORATORY MANUAL

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CLINICAL BACTERIOLOGY LABORATORY

LABORATORY MANUAL

General:

The purpose of the Bacteriology Lab section is to demonstrate and practice basic procedures

in diagnostic bacteriology, as well as currently used practical and rapid diagnostic tests and

kits. These will be applied for characterization of representative bacterial genera, and

identification of strains given under code.

Three teaching clusters are scheduled concomitant with the frontal lectures and discussions.

Each is comprised of two sessions. Participation in the Lab is compulsory. Duties include a

summarizing report for each cluster. In case of group experiments - all the results should be

reported.

Technical work will be performed in pairs. Wearing a lab coat is required for all sessions.

Bacteriology Lab 13/9/111

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Preparing a scientific report or manuscript

Preparing an outline is the most important step in the process of producing a report or a manuscript for

publication in a journal. The outline bears roughly the same relation to the final manuscript as an architectural

blueprint does to a finished house.

Its purpose is to divide the writing of the entire paper into a number of smaller tasks.

A good outline will organize the various topics and arguments in logical form. By ordering the topics you willidentify, before writing the manuscript, any gaps that might exist.

There is no single best way to prepare a scientific manuscript, except as determined by the individual writer

and the circumstances. You should know your own style of writing best. Whatever you decide to do, you

should follow at least these steps before beginning to write your manuscript.

Remember, at this stage, you are only constructing an outline. You are not writing; you just need to put down

some notes to guide your thinking.

1. Develop a central message of the manuscriptPrepare a central message sentence (20-25 words). If you were asked to summarize your paper in one

sentence, what would you say? Everything in the manuscript will be written to support this central

message.

2. Define the materials and methodsBriefly state the population in which you worked, the sampling method you employed, the materials you

used, and most importantly, the methods you used to carry out the study.

3. Summarize the question(s) and problem(s)

What was known before you started the study? What answers were needed to address the problem(s)? List

the key points pertaining to the question(s) and problem(s). What did you do to answer the question(s)?

4. Define the principal findings and results

Your central message sentence probably encapsulates the most important findings. There may be others

that you feel ought to be included. List these in note form. Don't worry about the order or about how manyyou put down.

5. Describe the conclusions and implications

Make brief notes on each of the implications that arise from your study. What are the principal conclusions

of your findings? What is new in your work and why does it matter? What are the limitations and the

implications of your results? Are there any changes in practice, approaches or techniques that you would

recommend?

6. Organize and group related ideas together

List each key point separately. Key points can be arranged chronologically, by order of importance or by

some other pattern. The organizing scheme should be clear and well structured. You can use a cluster map,

an issue tree, numbering, or some other organizational structure.

Identify the important details, describe the principal findings, and provide your analysis and conclusionsthat contribute to each key point.

7. Identify the references that pertain to each key point

8. Develop the introduction

Before beginning on the introduction, read through the notes you have made so far in your outline. Read

them through and see whether there is a coherent and cohesive story and a unifying theme that runs

through the outline.

Your introduction outline should start with the main message, describe what the purpose or objective of

your study was, how you went about doing the study, what you found and what are the implications of

what you found.

The purpose of the Introduction is to stimulate the reader's interest and to provide pertinent background

information necessary to understand the rest of the paper. You must summarize the problem to beaddressed, give background on the subject, discuss previous research on the topic, and explain exactly


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what the paper will address, why, and how. Besides motivating a reader to read your manuscript and to

care about your results, the Introduction is useful also to the journal's reviewers and editors in judging the

importance of your manuscript.

An Introduction is usually 300 to 500 words, but may be more, depending on the journal and the topic.

Therefore, the Introduction needs to be very concise, well structured, and inclusive of all the information

needed to follow the development of your findings.

Some people recommend that the Introduction be the first section written when writing a manuscript.Below are the steps in developing an effective Introduction. However, since every journal is different, it is

important that you look at papers in your targeted journal to determine whether they use all of these steps.

For example, some journals do not include conclusions in the Introduction.

1. Begin the Introduction by providing a concise backgroundaccount of the problem studied.

2. State the objectiveof the investigation. Your research objective is the most important part of the

introduction.

3. Establish the significanceof your work: Why was there a need to conduct the study?

4. Introduce the reader to the pertinent literature. Do not give a full history of the topic. Only quote

previous work having direct bearing on the present problem.

5. Clearly state your hypothesis, the variables investigated, and concisely summarize the methods

used.

6. Defineany abbreviations or specialized terms.

7. Provide a concise discussionof the results and findings of other studies so the reader understandsthe big picture.

8. Describe some of the majorfindingspresented in your manuscript and explain how they

contribute to the larger field of research.

9. State the principal conclusionsderived from your results.

10. Identify any questionsleft unanswered and any new questions generated by your study.

Other points to consider when writing your Introduction:

1. Be aware of who will be reading your manuscript and make sure the Introduction is directed to

that audience.

2. Move from general to specific: from the problem in the real world to the literature to your

research.3. Write in the present tense except for what you did or found, which should be in the past tense.

4. Be concise.

9. Result section

The purpose of a Results section is to present the keyresults of your research without

interpreting their meaning. It cannot be combined with the Discussion section unless the

journal combines the Results and Discussion into one section. The results should be

presented in an orderly sequence, using an outline as a guide for writing and following the

sequence of the Methods section upon which the results are based. For every result there

must be a method in the Methods section. It is important to carefully plan the tables and

figures to ensure that their sequencing tells a story. If you need help in preparing an outline

see our article Eight Steps to Developing an Effective Manuscript Outline at

www.sfedit.net/newsletters.html.

1. Determine which results to present by deciding which are relevant to the question(s)

presented in the Introduction irrespective of whether or not the results support the

hypothesis(es). The Results section does not need to include every result you

obtained or observed.

2. Organize the data in the Results section in either chronological order according to the

Methods or in order of most to least important. Within each paragraph, the order of

most to least important results should be followed.

3. Determine whether the data are best presented in the form of text, figures, graphs,or tables.

http://e2ma.net/go/298052955/222502/6428259/goto:http:/www.sfedit.net/newsletters.htmlhttp://e2ma.net/go/298052955/222502/6428259/goto:http:/www.sfedit.net/newsletters.html

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3. Summarize your findings and point the reader to the relevant data in the text, figures

and/or tables. The text should complement the figures or tables, not repeat the same

information.

4. Describe the results and data of the controls and include observations not presented

in a formal figure or table, if appropriate.

5. Provide a clear description of the magnitude of a response or difference. Ifappropriate, use percentage of change rather than exact data.

6. Make sure that the data are accurate and consistent throughout the manuscript.

7. Summarize the statistical analysis and report actual P values for all primary analyses.

8. Use the past tense when you refer to your results.

9. Number figures and tables consecutively in the same sequence they are first

mentioned in the text. Depending on the journal, they should be in order at the end

of the report after the References, or located appropriately within the text of your

results section.

10.Provide a heading for each figure and table. Depending on the journal the table titles

and figure legends should be listed separately or located above the table or below the

figure. Each figure and table must be sufficiently complete that it could stand on its

own, separate from the text.

11.Write with accuracy, brevity and clarity.

10. Discussion section

The purpose of the Discussion is to state your interpretations and opinions, explain the implications of your

findings, and make suggestions for future research. Its main function is to answer the questions posed in the

Introduction, explain how the results support the answers and, how the answers fit in with existing knowledge

on the topic. The Discussion is considered the heart of the paper and usually requires several writing attempts.

The organization of the Discussion is important. Before beginning you should try to develop an outline to

organize your thoughts in a logical form. You can use a cluster map, an issue tree, numbering, or some other

organizational structure. The steps listed below are intended to help you organize your thoughts.

To make your message clear, the discussion should be kept as short as possible while clearly and fully stating,supporting, explaining, and defending your answers and discussing other important and directly relevant

issues. Care must be taken to provide a commentary and not a reiteration of the results. Side issues should not

be included, as these tend to obscure the message. No paper is perfect; the key is to help the reader determine

what can be positively learned and what is more speculative.

1. Organize the Discussion from the specific to the general: your findings to the literature, to

theory, to practice.

2. Use the same key terms, the same verb tense (present tense), and the same point of view that

you used when posing the questions in the Introduction.

3. Begin by re-stating the hypothesis you were testing and answering the questions posed in the

introduction.

4.

Support the answers with the results. Address all the results relating to the questions,regardless of whether or not the findings were statistically significant.

5. Describe the patterns, principles, and relationships shown by each major finding/result and

put them in perspective. The sequencing of providing this information is important; first state

the answer, then the relevant results before citing the work of others. If necessary, point the

reader to a figure or table to enhance the story.

6. Support your answers by explaining how your results relate to expectations and to the

literature, clearly stating why they are acceptable and how they are consistent or fit in with

previously published knowledge on the topic.

7. Defend your answers, if necessary, by explaining both why your answer is satisfactory and

why others are not. Only by giving both sides to the argument can you make your

explanation convincing.

8. Discuss and evaluate conflicting explanations of the results. This is the sign of a good

discussion.


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9. Discuss any unexpected findings. When discussing an unexpected finding, begin the

paragraph with the finding and then describe it.

10. Identify potential limitations and weaknesses and comment on the relative importance of

these to your interpretation of the results and how they may affect the validity of the findings.

When identifying limitations and weaknesses, avoid using an apologetic tone.

11. Summarize concisely the principal implications of the findings regardless of statistical

significance.12. Provide recommendations (no more than two) for further research. Do not offer suggestions,

which could have been easily addressed within the study, as this shows there has been

inadequate examination and interpretation of the data.

13. Explain how the results and conclusions of this study are important and how they influence

our knowledge or understanding of the problem being examined.

14. Discuss everything, but be concise, brief, and specific in your writing of the Discussion.


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CLINICAL BACTERIOLOGY

LABORATORY

for


LABORATORY MANUAL

Lab #1

General Techniques

TEACHING UNIT: Bacteriology Lab 1

SUBJECT: General Techniques in Bacteriology Lab.


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Specific Subjects:

1. Collection and transport of clinical specimens.

2. Cultivation and isolation of bacteria.

3. Specialized Culture Media and Culture Conditions.4. Optical Methods for diagnosis

5. Testing antimicrobial susceptibility.

Introduction:

1. Collection and transport of clinical specimens

A number of collecting and transporting devices and media are available for medical

personnel to ensure that clinical specimens are collected and delivered to the

laboratory in the best condition possible. The nature and the design of the containers

are dictated by the type of specimen, the kind of examination anticipated and the time

required for transport to the laboratory.

Aerobic transport media are usually nonnutritive, phosphate buffered, and provide a

reduced environment due to sodium thioglycollate. Anaerobic transport media are

solidified with agar, thus inhibiting oxygen diffusion after the specimen on a swab is

inserted. Reducing agents in the medium combine with any free oxygen to maintain

anaerobiosis. Often, added Resazurin indicates the presence (pink) or absence (no

color) of oxygen in the medium.

The following devices will be on displayduring these sessions:Swabs, blood cultures bottles (aerobic, anaerobic, pediatric), various collection

containers.

2. Cultivation and Isolation of bacteria

2.1 Preparation of pure cultures by streaking plates.

The streak plate is used primarily for isolating microorganisms in pure

cultures from specimens containing a mixed flora. Obtaining isolated colonies

on plates permits a study of colonial morphology (see 4.1), and further

characterization methods, such as staining following fixation (see 4.2);streaking onto differential media (3.1), and testing for antimicrobial

susceptibility (see 5).

Inoculation of streak plates will be performed on various solid media, as

described in scheme 1, using disposable sterile quadriloops.


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2.2 Streaking for enumeration of microorganisms

Quantitative cultures are required for interpretation of urine cultures.

Calibrated loops (0.01 and 0.001 ml) will be utilized to inoculate bacteria

from simulated urine, as described in scheme 2.

3. Specialized Culture Media and Culture Conditions

Isolation of causative agent of disease depends on the use of appropriate culture

media and culturing (environmental conditions). Basic types of media and growth

conditions are introduced in the lab, beginning with the first session. Important terms

are: Supportive, Enrichment, Selective, and Differential media; Aerobic, Anaerobic,

Microaerophillic environments. (See more in Appendix 1).

Supportive media: Support the growth of a wide variety of microorganisms and

lacks inhibitory properties. These may be nonenriched or enriched with additives,

usually animal blood, in order to cultivate highly fastidious microbial species.

Examples: Non enriched: Nutrient Agar (NA), Trypticase Soy Agar (TSA), Brain

Heart Infusion (BHI) Agar

Enriched: Blood Agar(TSA with 5% sheep blood); Chocolate Agar.

Enrichment media: Broth media that encourage the growth of a few desired

microorganisms among large numbers of normal flora.

Example: Selenite broth is a peptone base broth containing sodium selenitetoxic for most Enterobacteriaceae, used for enrichment of isolation of

the smaller population of Salmonella and suppresses the growth of the

large numbers of normal stool organisms.

Selective media: Media containing inhibitory agent/s that select for certain

microorganisms to the disadvantage of others. Selective agents may be inhibitory

dyes or antibiotics, or components related to certain metabolic activities of the

organisms sought.

Examples: MacConkey agar selects against Gram-positive organisms due to

inclusion crystal violet and bile salts; Thayer Martin agar - blood

agar base enriched with hemoglobin and supplements selects for

Neisseria, and inhibits growth of other organisms by colistin, nystatin,

vancomycin, and trimethoprim.


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Differential media: Media employing factor/s that allow morphological distinction

of colonies of microorganisms possessing certain metabolic or cultural

characteristics.

Examples: Sheep Blood agar allow differentiation of hemolytic reactions ofvarious microorganisms. MacConkey agar differentiates lactose-

fermenting from lactose non-fermenting enteric bacilli.

Note: Blood agar is supportive for many microorganisms and allows

differentiation of hemolytic activity. MacConkey is selective against

Gram-positives and differentiates lactose fermentation of Gram-

negative enteric bacilli.

4. Optical Methods for diagnosis

4.1 Preliminary identification of colonies growing on solid media

Most clinical specimens are inoculated onto several solid media, including

some selective and differential media. The first clue for identification of an

isolated colony is understanding the nature of the medium on which the

organism is growing.

Colony characteristic morphology include: size (diameter); pigmentation of

the colony or the medium surrounding it, and texture (e.g. smooth, rough,

mucoid); hemolytic activity.

4.2 Differential staining of fixed material: Gram stain, Ziehl-Nielsen

The visualization of bacteria by means of staining procedures is an important

tool in identification and classification of isolates. Gram staining and Ziehl-

Nielsen (Acid-Fast stain) will be performed in the second session of this lab,

followed by microscopic examination.

5. Testing antimicrobial susceptibility of aerobic bacteria

The standardized procedure to test antimicrobial susceptibility of aerobic bacteria, is

based on diffusion of an antimicrobial agent from an impregnated paper disc throughthe agar gel. The organism to be tested is adjusted to a specific concentration and

uniformly spread over a solid agar plate, usually Muller-Hinton. After 16-18 hrs at

35C, the diameters of zones of inhibition produced around the discs are compared

with interpretive table to determine whether the organism is susceptible, intermediate

or resistant (Scheme 3).

In addition to performing this test, there will be a displayof MIC tests.


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Session No. 1 September 24th, 15:00-17:00 hours.

Microorganisms:

On nutrient agar (NA): Escherichia coli; Bacillus subtilis; Salmonella;

Staphylococcus coagulase negative.

On Chocolate agar: Haemophilus

In suspension: A mixture of two microorganisms.

A urine sample.

Materials:

5 NA plates - streaking for purified colonies;

2 NA plates - streaking with calibrated loops;

3 Blood Agar/ MacConkey plates; 1 Chocolate Agar - Culturing conditions

2 NA plates - testing antimicrobial susceptibility.

Antibiotics: Ampicllin 10 and 25 gr; Tetracycline, Chloramphenicol, Penicillin G,

Erythromycin.

2 Dipsticks - inoculating urine samples.

Saline or TSB.

Anaerobic jars.

Experimental:

1. Streaking for purified colonies:

Using the procedure described in Scheme 1, streak the following on five Nutrient

Agar(NA) plates: Escherichia coli, Bacillus subtilis, Salmonella; Staphyloccus

coagulase negative, and the mixed bacterial suspension.

Invert plates, and incubate overnight at 37C.

2. Streaking for enumeration of microorganisms in urine:

Using the procedure described in Scheme 2, streak from the urine sample two NA

plates as follows: one half of each plate with the 0.01 ml loop, and the second half -

with the 0.001 ml loop. Invert plates and incubate overnight at 37C.

3. Testing culturing media and conditions:

3.1 Blood/MacConkey plates- streak for purified colonies (scheme 1) two of the

identified microorganisms and the mixed suspension (each source on the two

halves). Invert plates and incubate overnight at 37C.

3.2 Streak Haemophilus on Chocolate agarplate using scheme 2.

Invert plates and incubate for 48 hrs at either (a) aerobic 37C, (b) 5%CO2

atmosphere 37C, or (c) anaerobic jar at 37C. (A group experiment!).


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4. Testing antimicrobial susceptibility

Test two of the identified microroganisms on 2 NAplates as follows (scheme 3):

4.1 Pick 4-5 purified colonies and suspend in 5 ml TSB (Tryptic Soy Broth) or

saline. Compare turbidity with a 0.5 McFarland Standard (Adjust asrequired).

4.2 Dip swab, wring out and inoculate the plate, spreading the suspension evenly.

4.3 Using a dispenser, apply antibiotic disks.

Invert plates and incubate overnight at 37C.

5. Inoculating urine samples using Dipsticks

Dip the plating sticks once in the urine suspension. Incubate overnight at 37C.


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Figure 1

Inoculation for colony isolation

Bacterium

1NABacterium

2

Bacterium

?

Quantitative inoculation

NA dipsticks1 l 10 l

Different Agar media

Blood /

MacConkey

Bacterial resistance examination

Bacterium 1 Bacterium 2 Bacterium ?

Bacterium Bacterium

21NA


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Session No. 2

Microorganisms:

Inoculated plates from Session 1.

Fixed mycobacterium for staining

Materials:

Gram stains

Ziehl-Neilsen stains

Experimental:

1. Examine plates for purified colonies, and evaluate your performance.

Calculate bacterial density in the urine sample.

2. Examine colonial morphology of bacteria streaked previously on all culture media.

3. Compare antimicrobial susceptibility of all microorganisms.

4. Perform Gram staining of two of the identified microorganisms, following their

fixation, according to instructions in Appendix 1.

5. Perform Ziehl-Neilsen staining of pre-fixed mycobacterium.

6. Examine all identified microorganisms under the microscope. Use the X100

magnification and immersion oil.

Summarize the morphological characteristics of the microorganisms examined in this

cluster:

Colonial morphology on the various media; microscopical morphology following

staining; and antimicrobial susceptibility.


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Results table for laboratory #1

Bacterium Colony

Morphology

(NA)

Blood

Agar

MacConkey

Agar

Single

bacterium

morphology

(Gramstain)

Antibiotic

Sensitivity /

Resistance

E. coli

Proteus

Staphylococcu

s

Bacillus

Bacteria Colony

Morphology

(NA)

Blood Agar MacConkey

Agar

Single bacterium

morphology

(Gram stain)

? 1 2 1 2 1 2 1 2


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Appendix 1: Culture media

Nutrient Agar

Nutrient Agar is used for the cultivation of bacteria and for the enumeration of organisms in

water, sewage, feces and other materials.

Early in the 20th century, the American Public Health Association (APHA) published the

formula for a general-purpose medium for growth of a wide variety of nonfastidious

microorganisms. This was in recognition of the need for a standardized medium for use in

the examination of water and wastewater, dairy products and various foods. This relatively

simple formulation has stood the test of time and, with the name of Nutrient Agar, is still

specified in current compendia of methods for the microbiological examination of a broad

spectrum of materials. Additionally, it is used in the laboratory for the cultivation and

maintenance of nonfastidious species.

Nutrient Agar consists of peptone, beef extract and agar. This relatively simple formulation

provides the nutrients necessary for the replication of a large number of microorganisms

which are not excessively fastidious. The beef extract contains water-soluble substances

including carbohydrates, vitamins, organic nitrogen compounds and salts. Peptones are the

principal sources of organic nitrogen, particularly amino acids and large-chained peptides.

Classical formula* per liter purified water:

Pancreatic Digest of Gelatin 5.0 g

Beef Extract 3.0

Agar 15.0

* Adjusted and/or supplemented as required to meet performance criteria. Final pH 6.80.2

Macconkey Agar

Macconkey Agar is a selective and differential medium for the detection of coliform and

enteric pathogens.

Macconkey developed one of the earliest culture media for the cultivation and identification

of enteric microorganisms. His medium included bile salts and lactose which aided in the

selection and differentiation of the enteric group.

The Macconkey Agar formulation has changed many times since it was originally

developed. BBL Macconkey Agar is the "classical" formula which has been in use for manyyears and is recommended in the United States Pharmacopeia (USP) for use in the

performance of Microbial Limit Tests. BBL prepared media in plates and tubes contain

Macconkey II agar, which is an improved modification specifically designed to increase the

inhibition of swarming Proteus species.

The concentration of bile salts in the medium is relatively low in comparison with other

enteric plating media; therefore, selectivity for gram-negative bacteria is not as great as in

some other formulations (e.g., Salmonella Shigella Agar and Hektoen Enteric Agar). Crystal

violet inhibits gram-positive microorganisms, especially enterococci and staphylococci.


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Differentiation of enteric microorganisms is achieved by the combination of lactose and

neutral red indicator. Colorless or pink to red colonies are produced depending upon the

ability of the isolate to ferment lactose.


Pancreatic Digest of Gelatin 17.0 gPancreatic Digest of Casein 1.5

Peptic Digest of Animal Tissue 1.5

Lactose 10.0

Bile Salts 1.5

Sodium Chloride 5.0

Neutral Red 0.03

Crystal Violet 0.001

Agar 13.5


Blood Agar

Trypticase Soy Agar, Modified (TSA II) supplemented with blood is used for cultivating

fastidious microorganisms and for the visualization of hemolytic reactions produced by

many bacterial species.

The nutritional compostion of Trypticase Soy Agar has made it a popular medium, both

unsupplemented and as a base for media containing blood. TSA II is an improved version of

the original Trypticase Soy Agar formulation for use with animal blood supplements. With 5

or 10% sheep blood, it is extensively used for the recovery and cultivation of fastidious

microbial species and for the determination of hemolytic reactions that are importantdifferentiating characteristics for bacteria, especially Streptococcus species. Some

investigators prefer the use of horse blood, but Trypticase Soy Agar with 5% Horse Blood is

not recommended for use with throat cultures.

The combination of casein and soy peptones renders the medium highly nutritious by

supplying organic nitrogen, particularly amino acids and longer-chained peptides. The

sodium chloride maintains osmotic equilibrium.

Defibrinated sheep blood is the most widely used blood for enriching agar base media.

Hemolytic reactions of streptococci are proper and growth of Haemophilushaemolyticus, a

nonpathogen whose hemolytic colonies are indistinguishable from those of beta-hemolyticstreptococci, is inhibited.

TSA II with 5% Sheep Blood provides excellent growth and beta-hemolysis by

Streptococcus pyogenes (Lancefield group A) and also provides excellent growth and

appropriate hemolytic reactions with other fastidious organisms. It is suitable for performing

the CAMP test for the presumptive identification of group B streptococci (S. agalactiae),

and for the use with low concentration (0.04 unit) bacitracin discs (Taxo A) for presumptive

identification of Group A streptococci (S. pyogenes). In addition to the medium in prepared

plates, tubed blood agar slants are provided for the growth and maintenance of stock cultures

of fastidious strains.


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The CAMP test, which is performed only on TSA II with 5% Sheep Blood, is based on the

formation of a zone of synergistic hemolysis at the junction of perpendicular streak inocula

of Staphylococcus aureus and group B streptococci. The reaction is caused by the

sphingomyelinase (beta-toxin) of S. aureus reacting with sphingomyelin in the sheep

erythrocyte membrane to produce ceramide. A nonenzymatic protein (CAMP protein),

produced by S. agalactiae, binds to the ceramide and leads to disorganization of the lipidbilayer of the sheep erythrocyte membrane resulting in complete lysis.

Approximate formula* per liter purified water:

Pancreatic Digest of Casein 14.5 g

Peptic Digest of Soybean Meal 5.0

Sodium Chloride 5.0

Agar 14.0

Growth Factors 1.5


Chocolate Agar

Chocolate Agar uses the same base as blood agar. Originally, red blood was added to the

molten base and the temperature raised enough to partially lyse the red blood cells (around

85 C), causing the medium to turn a chocolate-brown color. Now, hemoglobin and other

nutrients present in the lysed red cells, hemin (also known as "X" factor), and the coenzyme

nicotine adenine dinucleotide (called "V" factor) are added as supplements to a nutritionally

rich agar base. Neisseria gonorrhoeae and Haemophilus species, among other fastidious

organism, will grow best in the presence of the nutrients supplied by chocolate agar.

CLED Agar

CLED (Cystine-Lactose-Electrolyte-Deficient) Agar is used for the isolation, enumeration

and presumptive identification of microorganisms from urine.

CLED agar is recommended for use in plates or in urine dipsticks for detecting significant

bacteriuria by quantitative culture of urine. For relaiable results, inoculation of the medium

must occur as soon after collection as possible. Confluent or semiconfluent growth of

bacteria will occur on the surface of the dipstick medium when bacterial counts are greater

than 105 per ml of urine, as confirmed by plates inoculated by the calibrated-loop or

duplicate-dilution pour-plate methods.

Typical colonial morphology on CLED Agar as follows:

Escherichia coli Yellow colonies, opaque, center slightly deeper yellow

Klebsiella Yellow to whitish-blue colonies, extremely mucoid

Proteus Translucent blue colonies

Pseudomonas Green colonies, with typical matted surface and rough periphery

Enterococci Small yellow colonies, about 0.5 mm in diameter

S. aureus Deep yellow colonies, uniform in color

Staph. Coagulase (-) Pale yellow colonies, more opaque than S. faecalis


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Preparation of evenly coated plates:

For antibiotic resistance testing antibiotic impregnated filter paper disks, carrying a known amount of

antibiotics, are being placed onto an a freshly inoculated plates. The agent immediately begins to diffuse and

establish a concentration gradient around the paper disk. The highest concentration is closest to the disk. Upon

incubation the bacteria grow on the surface of the plate except where the antibiotic concentration in the

gradient around the disk is sufficiently high to inhibit growth. Following incubation the diameter of the zone

of inhibition is measured in millimeters.

Before disk placement, the plates surface is inoculated using a swab that has been submerged in bacterial

suspension standardized to match turbidity of 0.5 McFarland turbidity standard (i.e., 1.5x108CFU/ml). Thesurface of the plate is swabbed in three directions to ensure an even and complete distribution of the inoculum

over the entire plate.


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Appendix 3

Bacterial Colony Characteristics

Noting key features of bacterial colony is important for any bacterial identification. Criteria frequently used to

characterize bacterial growth include:

1. Colony size (usually measured in millimeters or described in relartive terms such as pinpoint, small, mediumlarge).

2. Colony pigmentation

3. Colony shape (includes form, elevation and margins of the colony smooth, rough)

4. Colony surface appearance (e.g., glistering, opaque, dull transparent)

5. Changes in agar media resulting from bacterial growth (e.g., hemolytic pattern on blood agar, changes in

color of pH indicator, pitting of the agar surface)

6. Odor (certain bacteria produce distinct odors that can be helpful in preliminary identification)

Appendix 4: The history of Gram stain and how it works

The Gram staining method, named after Hans Christian Gram, the Danish bacteriologist whooriginally devised this staining in 1882 (published 1884), is one of the most important

staining techniques in microbiology. It is almost always the first test performed for the

identification of bacteria. The primary stain of the Gram's method is crystal violet followed

by the addition of lugol. The microorganisms that retain the crystal violet-iodine complex

formed appear purple blue under microscopic examination. These microorganisms are

commonly classified as Gram-positive. Other bacteria that are not stained by crystal violet

and lugol are referred to as Gram negative, and appear red following a counter stain with

fuchsin/Safranin.

Gram staining is based on the ability of bacteria cell wall to retaining the crystal violet dye

which form complexes following the addition of lugol (Iodine salt) during organic solventtreatment. The cell walls for Gram-positive microorganisms have a higher peptidoglycan

content and lower lipid content than gram-negative bacteria. Bacteria cell walls are stained

by the crystal violet. Iodine is subsequently added to form the crystal violet-iodine complex

so that the dye cannot be removed easily. This step is commonly referred to as fixing the

dye. However, subsequent treatment with a decolorizer, which is a mixed solvent of ethanol

and acetone, dissolves the lipid layer from the gram-negative cells. The removal of the lipid

layer enhances the leaching of the primary stain from the cells into the surrounding solvent.

In contrast, the solvent dehydrates the thicker Gram-positive cell walls, and the cell wall

shrinks during dehydration. As a result, the diffusion of the violet-iodine complexes is

blocked, and the bacteria remain stained. The length of the decolorization is critical in

differentiating the gram-positive bacteria from the gram-negative bacteria. A prolongedexposure to the decolorizing agent will remove all the stain from both types of bacteria.

Some Gram-positive bacteria may lose the stain easily and therefore appear as a mixture of

Gram-positive and Gram-negative bacteria (Gram-variable).

Finally, a counterstain of basic fuchsin/Safranin is applied to the smear to give decolorized

gram-negative bacteria a pink color. Basic fuchsin stains many Gram-negative bacteria more

intensely than does safranin, making them easier to see. Some bacteria which are poorly

stained by safranin, such asHaemophilusspp.,Legionellaspp., and some anaerobic bacteria,

are readily stained by basic fuchsin, but not safranin. The polychromatic nature of the gram

stain enables determination of the size and shape of both Gram-negative and Gram-positive

bacteria. If desired, the slides can be permanently mounted and preserved for recordkeeping.


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Besides Gram's stain, there are a wide range of other staining methods available. By using

appropriate dyes, different parts of the bacteria structures such as capsules, flagella,

granules, and spores can be stained. Staining techniques are widely used to visualize those

components that are otherwise too difficult to see under a light microscope. In addition,

special stains can be used to visualize other microorganisms not readily visualized by the

Gram stain, such as mycobacteria, rickettsia, spirochetes, and others. In addition, there aremodifications of the Gram stain that allow morphologic analysis of eukaryotic cells in

clinical specimens.


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LABORATORY

for


LABORATORY MANUAL

Lab #2Gram-Positive Cocci


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SUBJECT: Gram-Positive Cocci: Staphylococci and Streptococci

Specific Subjects:

1. Performing laboratory tests leading to characterization and diagnosis of

Staphylococci and Streptococci.

2. Applying these tests on unknown samples.

3. Characterization of normal throat flora.

4. Examining a display of clinical specimens, including tests of antibiotic resistance,

and PYR tests.

Introduction:

Staphylococci and streptococci are gram-positive, nonsporeforming cocci, and significant

human pathogens. Staphylococci are part of the normal flora of human skin and the mucous

membranes, and as such will be taught later is this course. However, clinical isolates may be

cultured not only from exudate of skin infections or wound infections, but also from body

fluids or tissues during infections such as bacteremia, endocarditis, meningitis, or bacterial

pneumonia. Although staphylococci are not as fastidious as streptococci, suspected

specimens are primarily plated on blood-supplemented agar, and an important step in their

laboratory characterization is differentiating between the two genera. Thus, in this

laboratory, we will perform tests leading to characterization and diagnosis of Staphylococci

and Streptococci.

Basic information about staphylococci - virulence factors and diagnosis:

Staphylococci grown on agar media are arranged in clusters resembling clusters of grapes, as

implied from their Greek name. Organisms in clinical material are seen as single cells, pairs,

or even short chains. They are nonmotile, facultative anaerobic, catalase positive, and able

to grow in a medium containing 10% NaCl, and in a temperature range from 18 to 40 C.

Of the large number of staphylococcal species, three are commonly associated with human

diseases: S. aureus, S. epidermidis, and S. saprophyticus. S. aureus is distinguished from the

other two species due to its colonial morphology (yellow-white colonies, versus their whitecolonies), and its biochemical properties - hemolyticon blood agar, coagulase - positive,

and fermenting mannitol (both negative in theirs). Most laboratories do not characterize

further the coagulase negatives.

S. aureus is uniquely suited to be a human pathogen, due to its surface structure (e.g.

occasional capsule, Protein A, teichoic acids), and virulence factors (various types of toxins

- cytolytic toxins, exfoliative toxin, toxic shock syndrom toxin-1, enterotoxins, and enzymes

- coagulase, catalase, hyaluronidase, fibrinolysin, lipases, thermostable nucleases, and

penicillinases).


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Surface structures and their function: Capsules inhibit opsonization and phagocytosis, and

protect the bacteria from complement mediated lysis. Protein A is covalently linked to the

peptidoglycan layer, and binds the Fc receptor of IgG1, IgG2 and IgG4, thus inhibiting

opsonization and phagocytosis of S. aureus. Teichoic acidsare poor immunogens, and attach

the bacteria to mucosal surfaces, thus promoting their colonization.

Staphylococcal toxins: Cytolytic toxins: S. aureus produce at least five cytolytic toxins, also

described as hemolysins, but their activities are not restricted to red blood cells. These are

toxic to a variety of cells, including erythrocytes, leukocytes, and macrophages. Exfoliative

toxin or epidermolytic toxin, toxic shock syndrom toxin-1 and Enterotoxinsare associated

with specific syndromes.

Staphylococcal enzymes: Coagulase: S. aureus strains posses two types of coagulase - a

bound form, known as clumping factor and a free form. Coagulase bound to the cell wall can

directly convert fibrinogen into insoluble fibrin, and cause the staphylococci to clump

together. The cell-free coagulase interact with prothrombin into a thrombin-like substance,

which then converts fibrinogen into fibrin. Catalase is produced by all staphylococci. It

catalyzes the convertion of H2O2 into water and oxygen, thus protecting the bacteria against

H2O2 produced during bacterial metabolism or released following phagocytosis.

Hyaluronidase, fibrinolysin, and lipases facilitate the spread of S. aureus in connective

tissue, fibrin clots, and sebaceous areas of the body, respectively. Thermostable nucleasesof

S. aureus are phoshodiesterases with both endo-and exo-nucleolytic activity that cleaves DNA

and RNA.

Penicillinase: Most S. aureus strains (85 to 90%) are penicillin resistant, mainly due to -

lactamases (penicillinases) encoded by extrachromosomalplasmids. In addition, many strains

are also resistant to newer -lactamase-resistant semisynthetic penicillins such as methicillin,oxacillin, and nafcillin. Such strains are termed as Methicillin Resistant Staphylococcus aureus

or MRSA. This resistance is due partially to the presence of chromosomally encodedunusual

penicillin-binding proteins in their cell wall. Thus, identification of a MRSA by their

resistance to oxcaillin, ban therapy by -lactams, including cephalosporins.

Diagnostic tests and kits based on the following properties that will be performed or

displayed in the lab include: clumping factor, catalase test, Staphytect kit, and DNase

production.

Antibiotic susceptibility tests of S. aureus and MRSA will be displayed.

Streptococci and their diagnosis:

Most streptococci grow on standard laboratory media containing blood or blood products.

They are facultative anaerobes and grow well in 5 or 10% CO2 . Some strains grow better

under reduced oxygen tension, or microaerophillic conditions, e.g. in anaerobic jars with

activated GasPak (see Laboraotry No. 1). All are catalase-negative. Specifc -hemolytic

group A Streptococci strains produce extracellular enzymes, such as erythrogenic toxins,

streptolysins, DNases, hyalorunidase, and proteinases.


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In the clinical laboratory streptococci are usually differentiated according to their:

Morphology(colonies and by microscopy);

Hemolytic activityin blood agar plates (, or hemolysis);

Susceptibility to antibiotic, bacitracin(S. pyogenes i.e. -hemolytic Streptococcus group

A), to a detergent, optochin; (S. pneumoniae), or to bile salts(S. pneumoniae);Ability to hydrolyze Esculin (Enterococcus or S. faecalis), or PYR (L-pyrrolidonyl--

naphthylamide; Enterococcus and S. pyogenes).

In addition, their surface determinants may be classified into Lancefield groups by latexagglutination tests.

All of these tests will be practiced or displayed in the lab, using identified strains and

unknowns.

Rapid tests:

Note that following tests taught in this lab are performed within 30 min:

Clumping factor and catalase tests, PYR test, Staphytect kit and a streptococcal grouping kit.

These should be performed on isolated colonies, and require specific reagents. However, the

kit diagnosing Lancefield groups may be performed in the clinic, e.g. on throat swab

specimens, for a preliminary grouping of an infective Sterptococcus. This does not exclude

sending the specimen to a professional clinical laboratory!

Latex agglutination kits:

Two kits of this group will be introduced in this lab. The basic idea is that latex particles

coated with specific antibodies to microbial antigens will agglutinate microorganisms

bearing these antigens (Staphytect kit), or will be agglutinating when presented with

extracted microbial antigens (streptococcal grouping kit). Information about these kits is

presented in the Appendix to this lab.

Session No. 1

Microorganisms:


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On nutrient agar (NA): Escherichia coli; Staphylococcus coagulase negative;

Staphylococcus aureus (coagulase positive); MRSA.

On blood agar: Streptococcus -hemolyticgroup A;

-hemolytic: Streptococcus pneumoniae; andStreptococcus viridans;

Streptococcus faecalis (Enterococcus)

In suspension: An unknown (one of the above) under code.

Materials:

7 Blood agar plates - 1 for normal throat flora + 6 for the various strains as be described.

1 DNAse plate

3 Brain-heart infusion (BHI) plates containing 6.5% NaCl

1 Bile-Eschulin plate

4 test tubes with plasma

5 Bacitracin disks

3 Optochin disks

Hydrogen peroxide 3% for catalase test

Sterile swabs, Quadriloops, glass slides, sterile toothpicks.

Experimental:

Note before you begin streaking:

Staphylococci and E. coli are growing faster, produce larger colonies, and are less fastidious

than the streptococci. Thus identified streptococci and the relatively diluted unknown should

be applied in a gentle but dense streak, without turning the quadriloop.

1. Streaking normal throat flora.

Using the procedure described in page 1 of the Appendix, touch with a sterile swab

the pharynx or tonsils of your mate. Apply the specimen on a blood agar plate, andstreak for isolated colonies. Invert the plate and incubate overnight at 37C.


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2. Hemolytic activity of Staphylococci:

Use 3 Blood agar plates:Streak to get isolated colonies on halves of 2 plates:

E. coli, and the three identified Staphylococci. On a third plate streak the unknown on

two halves as noted above. Then, follow instructions on item 6. Finally, invert the

plates and incubate overnight at 37C.

3. Bacterial growth at high salt concentration:

Use 3 BHI + 6.5% NaCl plates- Streak in order to get isolated colonies on halves: E.

coli, S. aureus, Staphylococcus coagulase neagtive, the unknown, S. faecalis, and S.

viridans. Invert the plates and incubate overnight at 37C.

4. DNase production:

Use 1 DNase plate (details on the medium in the Appendix): - Using sterile

toothpicks apply a spot of each of E. coli, the three identified Staphylococci, and the

unknown. You may try and apply also the -hemolytic Streptococcus and another

streptococcus. Do not apply more than 6 strains per plate

Invert the plate and incubate overnight at 37C.

5. Hemolytic activity of Streptococci.

Use 3 Blood agar plates:On 2 plates, streak as noted above on page 5, each of the two-hemolytic streptococci (penumoniae and viridans) (two halves of the same plate for

each strain). On the third plate streak as noted above one half with S. faecalis, and one

half with -hemolytic Streptococcus. Then, follow instructions on item 6.

Finally, invert the plates and incubate overnight at 37C.

6. Susceptibility to Bacitracin and Optochin.

Use the 4 blood agar plates streaked with unknown (item 2) and streptococci (item

5) as follows:

Apply on one half of the two -hemolytic streptococci and of the unknown one

Bacitracin disk, and on the other half - one Optochin disc. Apply one Bacitracin disc on

each half of the plate streaked with S. faecalis, and -hemolytic Streptococcus.

7. Susceptibility to bile salt and Eschulin hydrolysis:

Use the Bile-Eschulin agarplate: Only bile-resistant and eschulin-hydrolyzing strain

will be able to grow on this plate. Eschulitine (product of Eschulin hydrolysis) will

interact with Fe++in the medium and blacken the agar.

Streak on halves S. faecalis, and the unknown. Invert the plate and incubate at 37C.8. Catalase test:


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On glass slides and using sterile toothpicks and H2O2, test all isolated strains

according to the procedure described in the Appendix. Record the reaction (positive

or negative). Try your unknown.

9. Clumping factor:

On glass slides and using plasma and sterile toothpicks, test all isolated strains

according to the procedure described in the Appendix. Record the reaction (positive

or negative). Try your unknown.


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Flow Chart

GAS

BE.

Coli

UnknownUnknown

O B

Strep

Pneumo

nia

Strep

Pneumo

nia

BO

Coa

Neg

Staph

Staph

Aureus

Normal

throat

flora

Strep

Viridans

O

Strep

Viridans

B

Blood AgarMRSAEnter

Faecalis

NaCl6.5%

Strepto

coccus

E.

Coli

MRSA Coa

Neg

Staph

Staph

AureusUnknown

Bile Esculin

AzideGAS Strep

Viridans

Enter

FaecalisStrep

Pneumo

nia

Unknown

Staph

AureusUnknownDNase Plate

Coa

Neg

Staph

Strepto

coccus

E.MRSAColi

Catalase

TestStaphylococcus

Streptococcus Unknown

Coagulase Test

Clumping FactorStaphylococcus Unknown

Staphylococcus & Unknown


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Lancefield Group

Streptococcus

Pyogenes

Enterococcus

Faecalis

Anti A

Anti D

Staphytect kit

Staph.

Aureus

MRSA Staph Coa.

Neg

Unknown

Staphytect

beads

Controlbeads

Gram Staining


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esults tableR

Blood Agar

)hemolysis(

NaCl

Agar

(growth)

BEA

,growt(

precipitat

)ion

Catalase

Coagulas

e

Clumpi

ng

Factor

Staphyt

-ect

DNase

Lancefield

group

Gram

Staining

S.

aureus

Staph

coa (-)

Strep.

pyogenes

Strep.

viridans

Strep.

pneu-

moniae

Entero-

coccus

faecalis

E. coli

Unknow

n


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Appendix 1: Taking throat swab specimens

Specimen collection and storageProper throat swab specimens MUST be collected in order to obtain accurate results. The

swabbing technique used to collect the specimen is as important to the test result as is the

performance of the test. The throat swab specimen must be collected under direct

visualization with good lighting. The pharynx and tonsils must be adequately exposed and

vigorously rubbed with a sterile rayon or dacron tipped swab. The tongue, uvula, and cheeks

must be avoided. Any exudate present should also be touched with the swab.

Immediately after collection, the swab should be delivered to the laboratory for testing. If

culture results are desired the swab may be streaked onto a blood agar plate before

performing Detect-A-Strep. The swab can be held in a Modified Stuart's transport system for

up to 48 hours after collection, but immediate testing is recomanded.


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Appendix 2: Procedures

Catalase test

Principle

The breakdown of hydrogen peroxide into oxygen and water is mediated by the enzyme

catalase. When a small amount of organisms that produce catalase is introduced into

hydrogen peroxide, rapid elaboration of bubbles of oxygen, the gaseous product of the

enzyme's activity, will be produced.

Method

1. With a loop or sterile wooden stick, transfer a small amount of pure growth from the

agar onto the surface of a clean, dry glass slide.

2. Immediately place a drop of 3% hydrogen peroxide (H2O2) onto a portion of a colony

on the slide.

3. Observe for the evolution of bubbles of gas, indicating a positive test.

CommentsIt has been recommended that the test be performed only on isolates grown on non-blood-

containing media. Although red blood cells contain some catalase, a technologist can

distinguish the very weak reaction of contaminating red blood cells by performing a control

slide catalase test with a small loopful of the blood-containing afar on the same slide of the

organism. If the catalase reaction from the colony is much stronger than that from the agar

alone, the test can be considered positive.


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Clumping factor ("slide coagulase") test

Principle

The presence of a cell surface-associated substance that binds fibrinogen and thus allows

aggregation of organisms in plasma containing fibrinogen is detected by observation ofclumping of cells.

Method

1. Place a drop of coagulase plasma (rabbit plasma with EDTA of citrate) on a clean,

dry glass slide.

2. Place a drop of distilled water of saline next to the drop of plasma as a control.

3. With a loop, straight wire, or wooden stick, emulsify an amount of the isolated

colony being tested in each drop, inoculating the water or saline first. Try to create a

smooth suspension.

4. Observe for clumping in the coagulase plasma drop and a smooth homogenous

suspension in the control. Clumping in both drops indicates that the organism

autoagglutinates and is unsuitable for the slide coagulase test.


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Appendix 3: Growth media

DNase test agarDNase test agar is used in detection of deoxyribonuclease (DNase) activity of bacteria and

fungi and especially as an aid for identification of pathogenic staphylococci.

DNase test agar with Toluidine Blue is used to aide in the differentiation and identification

of nonpigmented Serratia species isolated from clinical sources that may be incorrectly

identified asEnterobacterand Klebsiellaspecies.

The nutrients are supplies by the two peptones. The sodium-chloride maintains osmotic

equilibrium. The DNA enables the detection of DNase which depolymerizes the DNA,

resulting in the formation of a clear zone around the microbial growth when the plate is

flooded with hydrochloric acid. When toluidine blue O is incorporated into the medium or

when the plate is flooded with a 0.1% solution of the dye, DNase activity results in the

production of a visible bright rose-pink reaction due to the metachromatic property of the

toluidine blue.


Pancreatic digest of casein 15.0g

Papaic digest of soybean meal 5.0

Sodium chloride 5.0

Deoxyribonucleic acid 2.0

Agar 15.0


Bile esculin agar

Bile esculin agar is used to differentiate between group D streptococci and non-group D

streptococci.

Group D streptococci (including enterococci) hydrolyze the glycoside esculin to esculetin

and dextrose. Esculetin reacts with an iron salt to form a dark brown or black complex.

Ferric citrate is incorporated into the medium as an indicator of esculin hydrolysis and

resulting esculetin formation. Oxgall is used to inhibit gram-positive bacteria other thanenterococci.


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GlucoseEsculine

+

Esculetine

+ Fe++

Blackened agar

Approximate formula* per liter purified water

Pancreatic digest of gelatin 5.0g

Beef extract 3.0

Oxgall 20.0

Ferric citrate 0.5

Esculin 1.0

Agar 15.0



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Appendix 4: Commercial kits

Staphytect Plus kit

Intended use

Staphytect Plus is a latex slide agglutination test for the differentiation of staphylococci

which possess clumping factor, Protein A and certain capsular polysaccharides found in

MRSA strains from those which do not.

Principle of the test

Traditionally, differentiation between coagulase-positive and coagulase-negative

staphylococci has been performed either with the tube coagulase test that detects

extracellular staphylocoagulase or the slide coagulase test that detects the clumping factor

(bound coagulase) present on the bacterial cell surface. Several other differentiation tests are

also available including the passive haemagglutination test and the DNase test.

It has been reported that approximately 97% of human strains of Staphylococcus aureus

possess both bound coagulase and extracellular staphylocoaglulase. Protein A is found on

the cell surface of about 95% of human stains of S. aureusan has the ability to bind the Fc

portion of immunoglobulin G (IgG).

It has been observed that certain methicillin-resistant strains of S. aureus(MRSA) may

express undetectable levels of clumping factor and protein A. It has been shown however

that these strains all possess capsular polysaccharide. The capsule can mask both protein A

and the clumping factor thereby preventing agglutination.

Staphytect Plus uses blue latex particles coated with porcine fibrinogen and rabbit IgG

including specific polyclonal antibodies raised against capsular polysaccharides of S. aureus.When the reagent is mixed on a card with colonies of S. aureus, rapid agglutination occurs

through the reaction between (i) fibrinogen and clumping factor, (ii) Fc portion of IgG and

protein A, (iii) specific IgG and capsular polysaccharide. Agglutination may also accour

with other species which possess clumping factor or protein A such as Staphylococcus

hyicusand Staphylococcus intermedius. If neither clumping factor, protein A, nor specific

polysaccharide are present, agglutination will not occur and the result will be regarded as

negative. The most frequent coagulase and protein A negative isolates of staphylococci are

Staphylococcus epidermidis.

ClumpingFactor

CapsularPolysaccharides

Protein A

Fibrinogen

Latex

Bead

CapsularPolysaccharides

Staphylococcus

Aureus

Abs Capsu larPolysaccharides

Fc IgG


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Components of the kit

Staphytect Plus Test Reagentblue latex particles coated with both porcine fibrinogen and

rabbit IgG together with specific polyclonal antibodies raised against capsular

polysaccharide of S. aureus. Each kit contains sufficient reagent for 100 tests.

Streptococcal ("Lancefield" ) grouping kit ("Detect-A-Strep")

Material provided

Extraction reagents 1, 2, and 3

Ready-to-use reagents compounded for the efficient extraction of the group A carbohydrate.

Reagent 3 contains 0.1% sodium azide as preservative.

Detect-A-Strep Test Latex

Rabbit anti-Group A Streptococcus immunoglobulin-coated latex suspension containing0.1% sodium azide as preservative

Detect-A-Strep Control Latex

Normal rabbit gamma globulin-coated latex suspension containing 0.1% sodium azide as

preservative.

Detect-A-Strep Positive Control Antigen

Standardized suspension of non-viable group A streptococci which after extraction with

Reagents 1, 2, and 3 will produce moderate to strong agglutination with the Test Latex.

Detect-A-Strep Test Plate with six pairs of ceramic circles.


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Principles of the procedure

Detect-A-Strep is an easy to perform 2-step procedure:

1. Chemical extraction of the group A carbohydrate antigen.

2. Latex agglutination testing of the extract with a specific antibody-coated latex

suspension.

Within 5 minutes ready-to-use Extraction Reagents 1 and 2 extract the group A carbohydrateantigen from the streptococcal cell walls while the organisms are still on the swab. Adding

Extraction Reagent 3 stops the process and neutralizes the mixture sufficiently for the latex

agglutination step.

Using a specific antibody-coated latex suspension along with a control latex suspension, the

"extracted antigen" is tested for the presence of the specific group A carbohydrate. Visible

agglutination in the specific antibody-coated suspension in contrast to the milky smoothness

of the control suspension constitutes a positive test for the presence of group A streptococci

in the throat swab specime.

Reading results:

Compare the Test Latex circle with the Control Latex circle. The Control Latex should be

used as a standard for no agglutination; if it shows agglutination, the test is

UNITERPRETABLE and should be repeated. The Positive Control Antigen should show

moderate to strong agglutination with the Test Latex and no agglutination with the Control

Latex.

A NEGATIVE TEST shows no agglutination with either the Test or Control Latex

suspensions. Both suspensions should be smooth and milky, but some fine

graininess is normal.

A POSITIVE TEST shows DEFINITE AGGLUTINATION with the Test Latex

while the Control Latex remains distinctly smooth or "milky" by comparison.

After completing the test and recording the results, discard swabs and tubes and wash a drythe test plate.


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LABORATORY

for


LABORATORY MANUAL

Lab #3

Gram-Negative Bacilli


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SUBJECT: Gram-Negative Bacilli

Specific Subjects:

1. Performing laboratory tests to characterize and differentiate between members of the

Enterobacteriacea, Pseudomonas, andAcinetobacter.

2. Applying these tests on unknown samples.

3. Antimicrobial resistance

3.1 Analyzing antibacterial resistance due to beta-lactamases, using the rapid

nitrocefin test.

3.2 Examining a display of antimicrobial resistance to antimicrobials belonging

to different generations of development.

4. Characterization ofHaemophilusspecies by their requirement for growth-stimulating

factors (display).

5. Selective culturing conditions for isolation of Campylobacter from a mixed

population of gram-negatives (display).

Introduction

The familyEnterobacteriaceaeform the largest, most heterogeneous collection of medically

important gram-negative bacilli. These facultative anaerobicbacteria grow readily in vitro.

Specimens collected from normally sterile sources (spinal fluid; urine) can be inoculated

onto non-selective general purpose agar plates (e.g. blood agars). However, their cultivation

from specimens normally contaminated with other microorganisms (sputum, feces), requires

the use of selective media (e.g. MacConkey agar). The use of selective media containing

various sugars has the advantage of differentiating empirically lactose-fermenting species

from nonfermentingspecious. Examples used in this lab will be Salmonella-Shigella (SS)

Agar, Kligler Iron slants.

A battery of biochemical tests is available to analyze the metabolic properties of

Enterobactericeae, including production of specific enzymes (e.g. urease by Proteus,

tryptophane deaminase), type of fermentation pathways used and their end-products (e.g.

lactose fermentation by E. coli and Klebsiella, acetoin production by Klebsiella). In

addition to such selective & differentiating agars, we will use in this lab the API 20

identification kit. It represents a group of laboratory kits, each comprised of many

biochemical tests, that provide an analytical digitized profile of the cultured microorganisms,

and their accurate identification. Parallel kits are available for other groups of bacteria.


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In addition, tests that are used to differentiate between Enterobacteriacea and the gram-

negative opportunistic Pseudomonasand Acinetobacter, will be carried out. Among these -

tests of oxidase,and citrate utilization.

* To be able to make the most out of the tests performed in this lab, you should be able

to analyze characteristics of the following media: MacConkey (Appendix for Lab 1); SS

agar,Kligler Iron agar (Appendix for this lab).

(a) Which media components are the selective agents? Against which types of bacteria?

Which types of bacteria may grow?

(b) Which media components are the differentiating agents in the medium?

(clue: carbon sources)

(c) Which media components facilitate the identification of specific biochemical traits,

and how? (a clue for several media and traits - the specific pH indicators).

(d) Following bacterial growth, and resulting color pattern - characterize the profile of

the tested microorganisms.

* This should be prepared FOR the FIRST SESSION.

* Reading about metabolic pathways used by the microorganisms tested in this lab, and

their products is required. Please complete this preparation before the 2 nd session takes

place.

Several displays have been prepared for this lab:

(a) Characterization ofHaemophilusaccording to the requirement for factors X and V.

(What are these? In which rich medium these are not available to the bacteria, and in

which they are?)

(b) Antimicrobial susceptibility of all identified microorganisms in this laboratory.

We will use a collection of antimicrobials originating from different generations of

drug development, including recent ones:

Penicillin (P); Imipenem (IPM, a recent monobactam); Ciprofloxacin (Cip, quinolone),

Resprim (SXT, Sulphamethoxazole + Trimetoprim); Ceftazidime (CAZ, 3rd generation

Cephalosporin); (GM, aminoglycoside).

Which bacteria are highly susceptible to new drugs, and which are resistant to almost all?

(c) Selective isolation of Campylobacterfrom a background ofEscherichia coli:

An experimental simulation of a possible clinical situation.


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Finally - in the second session two importantrapid testswill be used:

(a) Oxidase- for identification of cytochrome oxidase containing bacteria;

(b) Nitrocefin or Cefinase test- for a rapid identification of beta-lactamase harboringbacteria, in order to avoid the use of penicillins or cephalosporins against resistant

strains.

See this appendices for detailed description.


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Session No. 1

Microorganisms:

On nutrient agar (NA): Escherichia coli; Salmonella; Proteus; Shigella; Klebsiella;

Pseudomonas; Acinetobacter

In suspension: An unknown (one of the above) under code.

Displays: Campylobacter; Haemophilus; Neisseria.

Antimicrobial resistance ofEscherichia coli; Proteus;


Materials:

4 MacConkey plates

4 SS agar plates

8 Kligler Iron agar slants

4 Simmons-Citrate agar tubes

4 Urea tubes

1 API test

5 ml sterile water

Quadriloops, glass slides, sterile toothpicks.

Experimental:

Note before you begin:

* You should be prepared (a) for analysis of media components, as pointed out in the

Introduction; (b) for profile of microorganisms growing on these media..

* Our goal is to get isolated colonies on the various plates, so be sure to streak these

properly. Follow your instructors in inoculating the tubes, the slants and the API

test.

1. Test the various bacteria using the various tests according to table 1.

Streak appropriate half plates for isolated colonies; Kligler Iron agar - streak on slantand stab the butt; Simmons-Citrate - stab the butt; Urea - inoculate and disperse a

colony. Incubate at 37C.

2. Inoculate your API kit with your unknown (dilute about 0.5 ml unknown with 4.5 ml

sterile water. Deliver aliquots into cupules, and proceed as described in the

Appendix.

Create a humid atmosphere (following your instructor). Incubate at 37C.

3. Displays: record results and summarize.


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Table 1

Tests performed in the 1stsession 2ndsession

Bacterium MacConkey

Plate

SS

Plat

e

Kligle

r

Iron

Slant

Simmons

Citrate

Tube

Urea API*

Kit

Test

Oxidase

Rapid

Test

Nitrocefin

Test

E. coli + + + + + + +

Salmonella + + + - + -

Proteus + + + + + -

Shigella + + + - - -

Klebsiella + + + - - -

Pseudomonas + + + + - + +

Acinetobacte

r

+ + + + - + +

Unknown + + + - + + - -

*API kit will be used for unknowns. In addition, there will be on display, a set of API kits

of all identified Enterobacteriaceae..


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Session No. 2

Microorganisms:

Psuedomonas, Acinetobacter, Escherichia coli.

Displays: Campylobacter; Haemophilus; Neisseria.

Antimicrobial resistance ofEscherichia coli; Proteus;


Materials:

(1) Oxidase test: Bacterial cytochrome oxidase will oxidize the substrate, impregnated in

a filter pad, into a colored product..

(2) Nitrocefin (Cefinase) test: This chromogenic cephalosporin changes its color upon

destruction of its beta-lactamic ring by beta-lactamase.

Experimental:

Follow your instructor:

(1) Use the oxidase test to compare Pseudomonas and Escherichia coli.

(2) Use the Nitrocefin test to compare Pseudomonas, Acinetobacter and Escherichia coli.

(3) Complete analysis of your unknowns in the API kit, by adding reagents for testing

TDA (tryptophane deaminase), IND (indole production), and VP (acetoin

production). Use the manufacturers tables for final identification.

(4) Go over all the results. Make sure (discuss with your instructor) that you can answer

all the questions.

In your report:

(1) Details about the selective-differentiating media used in this lab, as indicated in the

Introduction.

(2) A complete record of the results (growth, color of pH indicator, and the conclusion

about the bacterial metabolic traits).

(3) Same for your unknown + data obtained using the API kit, and identification of its

species.


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Flow Chart

MacConkey

Agar

E.coli Proteus

Shigella

Klebsiella Pseudomonas

AcinetobacterSalmonella Unknown

SS AgarE.coli Proteus Klebsiella Pseudomonas

Salmonella Shigella Acinetobacter Unknown

Simmons

CitrateE.coli Proteus Salmonella Shigella Klebsiella Pseudomonas

AcinetobacterUnknown

Acinetobacter

Urea

E.coli Proteus Salmonella Shigella Klebsiella Pseudomonas UnknownKligler

Proteus SalmonellaE.coli

Oxidase

Test

Pseudomonas AcinetobacterE.coli Unknown

Nitrocefin

Test

Pseudomonas AcinetobacterE.coli Unknown

Unknown

API Shigella Klebsiella UnknownE.coli Proteus Salmonella


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esults TableR

Test

Bacteria

NA SS Mac-

Conkey

Urea Simm

ons

Cit-

rate

API(TDA, VP ,Ind)

Oxida

se

Nitroc

efin

Suscep/

Res

E. coli

Klebsiella

Proteus

Shigella

Salmo-nella

Pseudo-

monas

Acineto-bacter

Unknown


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Appendix 1: Culture media

Salmonella-Shigella (SS) Agar

Intended useSalmonella-Shigella (SS) agar is a differentially selective medium for the isolation of

pathogenic eneteric bacilli; especially those belonging to the genus Salmonella. This

medium is not recommended for the primary isolation of Shigella.

Summary and explanation of the test

The culture media that have been developed for the selection and differentiation of enteric

microorganisms from clinical and nonclinical materials inhibit the growth of gram-positive

species to a varying degree due to the presence of either pure bile salts, mixtures of bile salts

or dyes. One of the formulations currently used in the plating of samples for detection of

enteric pathogens is Salmonella Shigella agar, which is an example for a media containing

bile salt mixtures. This medium is essentially a modification of the Desoxycholate-Citrate

agar.

Due to the relatively high level of selectivity, some Shigellastrains may not grow on SS agar

and, therefore, the medium is not recommended for the primary isolation of Shigella. Media

recommended for the isolation of Shigellaare Hektoen Enteric and XLD agars.


Salmonella Shigella agar is designated as a moderately selective medium based upon the

degree of inhibition of gram-negative microorganisms that it inhibits due to its content of

bile salts, brilliant green and citrates. Differentiation of enteric organisms is achieved by the

incorporation of lactose in the medium. Organisms that ferment lactose produce acid which,in the presence of the neutral red indicator, results in the formation of red colonies. Lactose

nonfermenters form colorless colonies. The latter group contains the majority of intestinal

pathogens, including Salmonellaand Shigella.

The sodium thiosulfate and ferric citrate enable the detection of hydrogen sulfide production

as evidenced by colonies with black centers.

Classical formula* per liter purified water

Beef extract 5.0 g

Pancreatic digest of caseain 2.5

Peptic digest of animal tissue 2.5

Lactose 10.0Bile salts 8.5

Sodium citrate 8.5

Sodium thiosulfate 8.5

Ferric citrate 1.0

Neutral red 0.025

Agar 13.5

Brilliant green 0.330 mg

* Adjusted and/or supplemented as required to meet performance criteria. pH 7.00.2

Directions for preparation from dehydrated product1. Suspend 60 g of powder in 1 L of purified water. Mix thoroughly.


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2. Gas production Some of the bacteria (E.colias an example) create gas during their

fermentation if we streak the bacteria correctly we may see this gas in the butt.

3. Utilization of sulfour bacteria that can use these molecules produce of H2S, which

binds to Fe ions creating a black precipitate.

4. Lactase All bacteria can utilize glucose, and they prefer to do so. So glucose is

fermented first (always). The fermentation of glucose in aerobic conditions does notproduce acidic products; In anaerobic conditions acid is produced and the media

changes yellow.

Lactose is used only be bacteria that are Lac positive. Lactose is utilized in aerobic

and anaerobic conditions, producing acid in both, also changing the media yellow.

Peptone may be utilized only in aerobic condition. The basic products cause a change

in the color of the media to red, which is of course seen only in the slant.

Order of carbohydrate utilization by bacteria:

Lac positive bacteria : GlucoseLactosePeptone

Lac negative bacteria : GlucosePeptone

The lactase property is decided by the color of the slant.

The anaerobic property is decided by the color of the butt.

Slant

Butt

Classical formula* per liter purified waterPancreatic digest of caseain 10.0 g

Peptic digest of animal tissue 10.0

Lactose 10.0

Dextrose 1.0

Sodium chloride 5.0

Ferric ammonium citrate 0.5

Sodium thiosulfate 0.5

Agar 15.0

Phenol red 0.025



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Expected results

After incubation, record the reaction in the slant and butt, noting gas formation and hydrogen

sulfide production.

Typical reactions produced by members of the Enterobacteriaceae (majority of species inthe particular genus) are presented below.

Slant Butt Gas H2S

Citrobacter Alkaline Acid + + or -

Edwardsiella Alkaline Acid + +

Escherichia coli Acid Acid + -

Enterobacter Acid* Acid + -

Morganella Alkaline Acid -

Proteus Alkaline or Acid Acid + +

Providencia Alkaline Acid -Salmonella Alkaline Acid + +

Shigella Alkaline Acid - -* Mat revert to alkaline even though lactose fermented (E. aerogenes).

CLED Agar

CLED (Cystine-Lactose-Electrolyte-Deficient) Agar is used for the isolation, enumeration

and presumptive identification of microorganisms from urine.

CLED agar is recommended for use in plates or in urine dipsticks for detecting significant

bacteriuria by quantitative culture of urine. For relaiable results, inoculation of the medium

must occur as soon after collection as possible. Confluent or semiconfluent growth of

bacteria will occur on the surface of the dipstick medium when bacterial counts are greater

than 105 per ml of urine, as confirmed by plates inoculated by the calibrated-loop or

duplicate-dilution pour-plate methods.

Typical colonial morphology on CLED Agar as follows:

Escherichia coli Yellow colonies, opaque, center slightly deeper yellow

Klebsiella Yellow to whitish-blue colonies, extremely mucoid

Proteus Translucent blue colonies

Pseudomonas Green colonies, with typical matted surface and rough periphery

Enterococci Small yellow colonies, about 0.5 mm in diameterS. aureus Deep yellow colonies, uniform in color

Staph. Coagulase (-) Pale yellow colonies, more opaque than S. faecalis

Simons-Citrate agar

Intended use

Simmons-Citrate agar is used for the differentiation of gram-negative bacteria on the basis of

citrate utilization.

Summary and explanation of the test


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Koser, in 1923, developed a liquid medium consisting of inorganic salts in which an

ammonium salt was the only source of nitrogen and citrate was the sole carbon source in

order to differentiate between what are now known as Escherichia coli and Enterobacter

aerogenes as part of the IMViC (Indole-Methyl Red-Voges Proskauer-Citrate) reactions.

Simmons, in 1926, modified Koser's formulation with the addition of 1.5% agar and

bromthymol blue. Organisms capable of metabolizing citrate grow well on this medium.


Organisms able to utilize ammonium dihydrogen phosphate and sodium citrate as the sole

sources of nitrogen and carbon respectively will grow on this medium and produce an

alkaline reaction as evidenced by a change in color of the bromthymol blue indicator from

green (neutral) to blue (alkaline).

Classical formula* per liter purified water

Ammonium dihydrogen phosphate 1.0 g

Dipotassium phosphate 1.0

Sodium chloride 5.0

Sodium citrate 2.0

Magnesium sulfate 0.2

Agar 15.0

Bromthymol blue 0.08


DrySlide Nitrocefin (-lactamase test)

Intended use

DrySlide Nitrocefin is used in detecting beta-lactamase production by bacteria, particularly

Neisseria gonorhoeae, Haemophilus influenzae Moraxella catarrhalis, Staphylococcus

species, and anaerobic bacteria.

Summary and explanation

The beta-lactamase test is a qualitative procedure for detecting the production of beta-

lactamase by bacteria. The reaction is based on the production of a colored compound when

the substrate, nitrocefin, is exposed to beta-lactamase producing culture.

Principles of the procedureThe enzyme, beta-lactamase, originally described by Abraham and Chain, is produced by

various organisms and is a mechanism of their resistance to penicillins and cephalosporins.

Test methods used to detect beta-lactamase include iodometric and chromogenic

cephalosporin procedures.

Some staphylococci may require induction (exposure to a beta-lactam agent) to increase

production of beta-lactamase to detectable levels. According to standard references, any

negative beta-lactamase test result from a non-induced staphylococcal isolate, regardless of

the test method, should be confirmed by induction of the isolate and repeat testing.

DrySlide Nitrocefin emplys nitrocefin, a cephalosporin compound first described by Glaxo

Research, in the chromogenic cephalosporin test methodology. If beta-lactamase is produced

by a culture, it hydrolyzes the beta-lactam ring of nitrocefin, producing cephalosporanic


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acid. A distinctive color change is associated with this reaction wherein the pale yellow

nitrocefin turns pink after hydrolysis.

Both aerobic and anaerobic beta-lactamase-producing (i.e., beta-lactamase-positive) bacteria

effect this color change; organisms not producing beta-lactamase do not alter the pale yellow

color of nitrocefin within the same limits of the test.

Results

Beta-lactamase-positive organisms change the color of the reaction area from yellow to pink.

A positive result will develop within 5 minutes for most bacterial strains. However, positive

reactions may take up to 60 minutes to develop for some staphylococci.

Bet

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Lab Manual 2012-13