Lab Manual 2012-13

Medical School for International Health
CLINICAL
BACTERIOLOGY
LABORATORY
for
Microbiology 2012-2013
LABORATORY MANUAL
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.
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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 will
identify, 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 manuscript

Prepare 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 methods

Briefly 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 many
you 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 conclusions
that contribute to each key point.
7.
8.
Identify the references that pertain to each key point

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 be
addressed, 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 background account of the problem studied.
2. State the objective of the investigation. Your research objective is the most important part of the
introduction.
3. Establish the significance of 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. Define any abbreviations or specialized terms.
7. Provide a concise discussion of the results and findings of other studies so the reader understands
the big picture.
8. Describe some of the major findings presented in your manuscript and explain how they
contribute to the larger field of research.
9. State the principal conclusions derived from your results.
10. Identify any questions left 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 key results 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.
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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. If
appropriate, 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.
2.
3.
4.
5.
6.
7.
8.
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Organize the Discussion from the specific to the general: your findings to the literature, to
theory, to practice.
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.
Begin by re-stating the hypothesis you were testing and answering the questions posed in the
introduction.
Support the answers with the results. Address all the results relating to the questions,
regardless of whether or not the findings were statistically significant.
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.
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.
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.
Discuss and evaluate conflicting explanations of the results. This is the sign of a good
discussion.
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9.
10.
11.
12.
13.
14.
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Discuss any unexpected findings. When discussing an unexpected finding, begin the
paragraph with the finding and then describe it.
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.
Summarize concisely the principal implications of the findings regardless of statistical
significance.
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.
Explain how the results and conclusions of this study are important and how they influence
our knowledge or understanding of the problem being examined.
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:
Bacteriology Lab
General Techniques in Bacteriology Lab.
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Specific Subjects:
1.
2.
3.
4.
5.
Collection and transport of clinical specimens.

Cultivation and isolation of bacteria.
Specialized Culture Media and Culture Conditions.
Optical Methods for diagnosis
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 display during 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 selenite

toxic 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:
Bacteriology Lab
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.
4.
Examples:
Sheep Blood agar allow differentiation of hemolytic reactions of

various microorganisms. MacConkey agar differentiates lactosefermenting 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 Gramnegative enteric bacilli.
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 ZiehlNielsen (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 through
the 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 display of 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 agar plate 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 NA plates 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 as
required).
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
NA
Bacterium
2
Bacterium
1
Bacterium
?
Quantitative inoculation
NA
10 l
1 l
dipsticks
Different Agar media

Blood /
MacConkey
Bacterium 1
Bacterium 2
Bacterium ?
Bacterial resistance examination
NA
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Bacterium
1
Bacterium
2
12
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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
(Gram
stain)
Antibiotic
Sensitivity /
Resistance
E. coli
Proteus
Staphylococcu
s
Bacillus
Bacteria Colony
Morphology
(NA)
?
1
2
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Blood Agar
MacConkey
Agar
14
Single bacterium
morphology
(Gram stain)
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
Beef Extract
Agar
5.0 g
3.0
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 many
years 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
Pancreatic Digest of Casein
Peptic Digest of Animal Tissue
Lactose
Bile Salts
Sodium Chloride
Neutral Red
Crystal Violet
Agar
17.0 g
1.5
1.5
10.0
1.5
5.0
0.03
0.001
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 important
differentiating 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 Haemophilus haemolyticus, a
nonpathogen whose hemolytic colonies are indistinguishable from those of beta-hemolytic
streptococci, 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 lipid
bilayer of the sheep erythrocyte membrane resulting in complete lysis.
Approximate formula* per liter purified water:
Pancreatic Digest of Casein
Peptic Digest of Soybean Meal
Sodium Chloride
Agar
Growth Factors
14.5 g
5.0
5.0
14.0
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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Appendix 2: Bacterial Cultivation

Isolation of bacteria from specimens
The basic flow of procedures involved in the laboratory diagnosis of infectious disease is:
1. Direct examination of patient specimens for the presence of etiological agents.
2. Growth and cultivation of the agents from these specimens.
3. Analysis of the cultivated organisms to establish their identification and other pertinent characteristics such
as susceptibility to antimicrobial agents.
The process of bacterial cultivation involves the use of optimal artificial media and incubation conditions to
isolate and identify the bacterial etiologies of an infection as rapidly and as accurately as possible.
To enhance the growth, isolation and selection of etiological agents, specimen inocula are usually spread over
the surface of plates in a standard pattern so that individual bacterial colonies are obtained and semiquantitative analysis can be performed. A commonly used streaking technique involves the spreading of the
bacteria into 4 quarters on the plate:
1. Touch the specimen or a bacterial colony with the bacteriological needle (4 sided needle).
2. Draw three parallel lines
3. Turn the needle and the plate 90o
4. Draw three parallel line, touching at the edge of the lines drawn before
5. Turn the needle and the plate 90o
6. Repeat steps 4 and five two additional times
Turn 90
Turn 90
Turn 90
Quantitative plating:
The aim of quantitative plating is to estimate the number of Colony Forming Units (CFU) in a specimen for
example in a urine sample.
In the lab you will find two types of calibrated bacteriological loops one that measures 1l and one that
measures 10 l.
Method of plating: Insert the bacteriological loop into the urine specimen. Touch at the middle of the plate,
then draw a line in the middle of the plate. In the next step the dispense the bacteria by moving through the line
as many time as possible, to dilute the bacteria on the plate and obtain single colonies.
Perform the plating once the 1l and on a second plate with the 10 l loop.
On the next day check your plates and count the number of colonies and calculate the number of colonies /ml
of urine.
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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). The
surface 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, medium
large).
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 who
originally 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 solvent
treatment. 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 prolonged
exposure 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 as Haemophilus spp., Legionella spp., 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 record
keeping.
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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 are
modifications of the Gram stain that allow morphologic analysis of eukaryotic cells in
clinical specimens.
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LABORATORY
for
LABORATORY MANUAL
Lab #2
Gram-Positive Cocci
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SUBJECT:
Gram-Positive Cocci : Staphylococci and Streptococci
Specific Subjects:
1.
2.
3.
4.
Performing laboratory tests leading to characterization and diagnosis of

Staphylococci and Streptococci.
Applying these tests on unknown samples.
Characterization of normal throat flora.
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 white
colonies), and its biochemical properties - hemolytic on 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 acids are 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 Enterotoxins are 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 nucleases of
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 extrachromosomal plasmids. 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 encoded unusual
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 activity in 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 latex
agglutination 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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Escherichia coli; Staphylococcus coagulase negative;

Staphylococcus aureus (coagulase positive); MRSA.
On blood agar:
Streptococcus -hemolytic group A;

-hemolytic: Streptococcus pneumoniae; and
Streptococcus 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, and
streak 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:
8.
Use the Bile-Eschulin agar plate: 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.
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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Session No. 2: (four hours).

Materials:
HCl 1N - to display results of the DNase test.
Staphytect kit
Streptococcal grouping kit
Gram stains
Experimental:
In this session you will record the growth and appearance of colonies of the various strains
on the plates, stain them with Gram stains, finish analysis of the DNase test, and perform the
two latex agglutination tests. You will be able to identify your unknown. You should be able
to answer questions from the lab teaching unit.
A display of the PYR test and antimicrobial susceptibility of these microorganisms will
be on. In addition, reference plates will be available .
1.
Complete the DNase test:

Pour HCl 1N on the DNase plate. Using a dark background, watch for appearance of
clear zones surrounding DNase producing bacteria.
2.
Record growth, hemolysis and susceptibility results.
3.
Gram-stain two streptococci , two staphylococci, and your unknown, according to the
procedure described in Lab 1. Inspect bacterial morphology under the microscope.
Make sure that you inspect all identified bacteria and your unknown.
4.
Staphytect kit:
Follow instructions in the Appendix and test all identified staphylococci. If your
unknown is a staphylococcus you may try that as well.
5.
Streptococcal grouping kit.

Follow instructions in the Appendix and text only the -hemolytic, the S. faecalis.
After preparing the extract, continue with the following combinations:
-hemolytic
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S. faecalis
Anti group A
Anti group D
29
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Flow Chart
Enter GAS
Faecalis B
E. MRSA
Coli
Strep
Pneumo
nia
O
Unknown Unknown
O B
Strep
Pneumo
nia
B
Coa Staph
Neg Aureus
Staph
Blood Agar
Normal
throat
flora
Strep Strep
Viridans Viridans
O B
NaCl 6.5%
Strepto
coccus
E. MRSA
Coli
Unknown
GAS Enter
Faecalis
Unknown
Unknown
DNase Plate
Coa
Neg
Staph
MRSA
Staphylococcus
Streptococcus
Staphylococcus
Unknown
Catalase
Test
Coagulase Test
Staphylococcus & Unknown
Bacteriology Lab
Bile Esculin
Azide
Strep Strep
Pneumo Viridans
nia
Staph
Aureus
Strepto
coccus
E.
Coli
Coa Staph
Neg Aureus
Staph
Unknown
30
Clumping Factor
13/9/11
Lancefield Group
Streptococcus
Pyogenes
Enterococcus
Faecalis
Anti A
Anti D
Staphytect kit
Staph.
Aureus
MRSA
Staph Coa.
Neg
Unknown
Staphytect
beads
Control
beads
Gram Staining
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Results table
Blood Agar
(hemolysis)
NaCl
Agar
(growth)
BEA
(growt,
precipitat
ion)
Catalase
Coagulas
e
Clumpi
ng
Factor
Staphyt
-ect
DNase
Lancefield
group
S.
aureus
Staph
coa (-)
Strep.
pyogenes
Strep.
viridans
Strep.
pneumoniae
Enterococcus
faecalis
E. coli
Unknow
n
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Gram
Staining
Appendix 1: Taking throat swab specimens

Specimen collection and storage
Proper 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.
Comments
It has been recommended that the test be performed only on isolates grown on non-bloodcontaining 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 of
clumping 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 agar
DNase 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 as Enterobacter and Klebsiella species.
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
Papaic digest of soybean meal
Sodium chloride
Deoxyribonucleic acid
Agar
15.0g
5.0
5.0
2.0
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 than
enterococci.
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+
Esculine
Glucose
Esculetine
+ Fe++
Blackened agar
Approximate formula* per liter purified water
Pancreatic digest of gelatin
Beef extract
Oxgall
Ferric citrate
Esculin
Agar
5.0g
3.0
20.0
0.5
1.0
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. aureus an 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
hyicus and 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.
Latex
Bead
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Fibrinogen
Clumping
Factor
Fc IgG
Protein A
Abs Capsular
Polysaccharides
Capsular
Polysaccharides
38
Staphylococcus
Aureus
13/9/11
Components of the kit

Staphytect Plus Test Reagent blue 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 containing
0.1% 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 carbohydrate
antigen 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 dry
the 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, and Acinetobacter.
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 of Haemophilus species 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 family Enterobacteriaceae form the largest, most heterogeneous collection of medically
important gram-negative bacilli. These facultative anaerobic bacteria 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 nonfermenting specious. 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 gramnegative opportunistic Pseudomonas and 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 of Haemophilus according 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 Campylobacter from a background of Escherichia coli:

An experimental simulation of a possible clinical situation.
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Finally - in the second session two important rapid tests will be used:
(a)
Oxidase - for identification of cytochrome oxidase containing bacteria;
(b)
Nitrocefin or Cefinase test - for a rapid identification of beta-lactamase harboring

bacteria, 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:
Escherichia coli; Salmonella; Proteus; Shigella; Klebsiella;

Pseudomonas; Acinetobacter
In suspension:
An unknown (one of the above) under code.
Displays:
Campylobacter; Haemophilus; Neisseria.

Antimicrobial resistance of Escherichia 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 slant
and 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 1st session
Bacterium
2nd session
MacConkey
SS
Kligle
Simmons Urea
API*
Oxidase Nitrocefin
Plate
Plat
Citrate
Kit
Rapid
Iron
Tube
Test
Test
Test
Slant
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 of Escherichia 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
SS Agar
Kligler
Simmons
Citrate
Urea
E.coli
E.coli
E.coli
E.coli
E.coli
Proteus
Salmonella
Shigella
Klebsiella Pseudomonas
Acinetobacter Unknown
Salmonella
Shigella
Acinetobacter Unknown
Proteus
Proteus
Proteus
Proteus
Salmonella
Salmonella
Shigella
Shigella
Acinetobacter
Acinetobacter
Unknown
Unknown
Salmonella
Unknown
Oxidase
Test
E.coli
Pseudomonas Acinetobacter Unknown
Nitrocefin
Test
E.coli
Pseudomonas Acinetobacter
API
Bacteriology Lab
E.coli
Proteus
Salmonella
48
Shigella
Klebsiella
Unknown
Unknown
13/9/11
Results Table
Test
NA
SS
MacConkey
Urea
Bacteria
E. coli
Simm
ons
Citrate
API
(TDA, VP ,Ind)
Oxida
se
Nitroc Suscep/
efin
Res
Klebsiella
Proteus
Shigella
Salmonella
Pseudomonas
Acinetobacter
Unknown
Bacteriology Lab
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Appendix 1: Culture media

Salmonella-Shigella (SS) Agar
Intended use
Salmonella-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 Shigella strains 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 Shigella are 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 Salmonella and 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
10.0
Bile 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 product

1. Suspend 60 g of powder in 1 L of purified water. Mix thoroughly.
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2. Heat with frequent agitation an boil for 1 min to completely dissolve the powder.
3. Cool the medium to approximately 45 to 50C and pour into Petri dishes using
approximately 20 ml per plate.
4. Allow the plates to dry for approximately 2 h with the covers partially removed. DO
NOT AUTOCLAVE.
5. Test samples of the finished product for performance using stable, typical control
cultures.
Kilgler Iron agar
Intended use
Kligler Iron agar is used for differentiation of members of the Enterobacteriaceae on the
basis of their ability to ferment dextrose and lactose and to liberate sulfides.
Summery and explanation of the test
In 1911, Russell described a new double sugar tube medium for the isolation of typhoid
bacilli from urine and feces. Six years later, Kligler developed a simple lead acetate medium
for the differentiation of the typhoid-paratyphoid group. Subsequently, Kligler evaluated
culture media used in the isolation and differentiation of typhoid, dysentery, and allies bacilli
and endorsed Russell's medium. Bailey and Lacey substituted phenol red for the Andrade
indicator previously use as a pH indicator.
The current formulation of Kligler Iron agar combines features of Kligler's lead acetate
medium with those of Russell's double sugar agar.
Principle of the procedure
Kligler Iron agar, in addition to casein and meat peptones, contains lactose and dextrose
which enable the differentiation of species of enteric bacilli due to color changes of the
phenol red pH indicator in response to the acid produced during the fermentation of these
sugars. The dextrose concentration is only 10% of the lactose concentration. The
combination of ferric ammonium citrate and sodium citrate enables the detection of
hydrogen sulfide production.
Lactose nonfermenters (e.g., Salmonella and Shigella) initially produce a yellow slant due to
acid produced by the fermentation of the small amount of dextrose. When the dextrose
supply is exhausted in the aerobic environment of the slant, the reaction reverts to alkaline
(red slant) due to oxidation of the acids. The reversion does not occur in the anaerobic
environment in the butt, which remains acid (yellow butt). Lactose fermenters produce
yellow slants and butts because enough acid is produced in the slant to maintain an acid pH
under aerobic conditions. Organisms incapable of fermenting either carbohydrate produce
red slants and butts.
Hydrogen sulfide production is evidenced by a black color either throughout the butt, or in a
ring formation near the top of the butt. Gas production (aerogenic reaction) is detected as
individual bubbles or by splitting or displacement of the agar.
Kligler Media helps us learn about 4 important properties.
1. Ability to live in anaerobic conditions bacteria that can live in anaerobic
conditions, do so in the butt. They ferment glucose and/or lactose, producing acidic
products which change the media (in the butt part) to yellow.
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2. Gas production Some of the bacteria (E.coli as 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 not
produce 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 : Glucose Lactose Peptone
Lac negative bacteria : Glucose Peptone
The lactase property is decided by the color of the slant.
The anaerobic property is decided by the color of the butt.
Slant
Butt

Pancreatic 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 in
the particular genus) are presented below.
Citrobacter
Edwardsiella
Escherichia coli
Enterobacter
Morganella
Proteus
Providencia
Salmonella
Shigella
Slant
Alkaline
Alkaline
Acid
Acid*
Alkaline
Alkaline or Acid
Alkaline
Alkaline
Alkaline
Butt
Acid
Acid
Acid
Acid
Acid
Acid
Acid
Acid
Acid
Gas
+
+
+
+
+
-
H2S
+ or +
+
+
-
* 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 diameter
S. 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).
Ammonium dihydrogen phosphate
Dipotassium phosphate
Sodium chloride
Sodium citrate
Magnesium sulfate
Agar
Bromthymol blue
1.0 g
1.0
5.0
2.0
0.2
15.0
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 betalactamase by bacteria. The reaction is based on the production of a colored compound when
the substrate, nitrocefin, is exposed to beta-lactamase producing culture.
The 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
Bacteriology Lab
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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.
Beta-lactamase-negative organisms do not change the color of the reaction area.
Bacteriology Lab
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Appendix 2: Other procedures

API 20
API 20 strip consists of 20 microtubes containing dehydrated substrates. These tests are
inoculated with a bacterial suspension which reconstitutes the media. During incubation,
metabolism produces color changes that are either spontaneous or revealed by the addition
of reagents.
The reactions are read according to the interpretation Table and the identification Table, the
Analytical Profile Index or the identification software.
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Spot oxidase test (Kovac's Method)

Principle
The cytochrome oxidase enzyme is able to oxidize the substate tetramethyl-pphenylenediamine dihydrochloride, forming a colored end product, indophenol. The dark
purple end product will be visible if a small amount of growth from a strain that produces the
enzyme is rubbed on substrate-impregnated filter paper.
Method
1. Prepare a solution of 1% tetramethyl-p-phenylenediamine dihydrochlorate in sterile
distilled water each day. To eliminate some work, 50 mg reagent powder can be
weighted into each of a large number of plastic, snap-top tubes at one sitting. Each
day of use, 5 ml of water is added to one tube (to a predrawn line, for example)
which is discarded after that day's use. The reagent is also commercially produced in
individually sealed glass ampules for daily use.
2. Place a filter paper circle into a sterile plastic disposable Petri dish and moisten the
filter paper with several drops of the fresh reagent.
3. Remove a small portion of the colony to be tested (preferably not more than 24 h old)
with a platinum wire or wooden stick and rub the growth on the moistened filter
paper.
4. Observe for a color change to blue or purple within 10 s (timing is critical.
Expected results
Positive organisms such as Neisseria species will turn the filter paper dark purple within 10
seconds; negative organism material such as that from E. coli will remain colorless or the
color of the colony within 10 seconds.
Urease test
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The test is used to differentiate urease-positive Proteus species from others members of the
Enterobacteriaceae. Some strains of Enterobacter and Klebsiella species are also ureasepositive. The test may be used to distinguish Psychrobacter phenylpyruvicus from Moraxella
species. Corynebacterium diphtheriae is urease-negative which differentiates it from the
urease-positive Corynebacterium ulcerans and Corynebacterium pseudotuberculosis.
Helicobacter pylori also possesses the ability to split urea. Most Brucella species are ureasepositive. Bacteroides ureolyticus splits urea rapidly, usually within a few minutes, and is a
quick way of identifying this organism. The urease test may aid in the identification of
Cryptococcus species which produces a positive result after prolonged incubation
Test principle
Some bacteria are able to produce the enzyme urease that attacks the nitrogen and carbon
bond in amide compounds such as urea, forming the end products ammonia, CO2, and water.
Urease activity (the urease test) is detected by growing bacteria in a medium containing urea
and using a pH indicator such as phenol red. When urea is hydrolyzed, ammonia
accumulates in the medium and makes it alkaline. This increase in pH causes the indicator to
change from orange-red to deep pink or purplish red (cerise) and is a positive test for
urea hydrolysis. Failure of a deep pink color to develop is a negative test.
Medical Application
In the clinical laboratory, members of the genus Proteus can be distinguished from other
enteric nonlactose-fermenting bacteria (Salmonella, Shigella) by their fast urease activity.
P. mirabilis is a major cause of human urinary tract infections.
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Bacteriology Lab
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Medical School for International Health

Medical School for International Health

CLINICAL BACTERIOLOGY LABORATORY

Preparing a scientific report or manuscript

Develop a central message of the manuscript

Define the materials and methods

Summarize the question(s) and problem(s)

Define the principal findings and results

Describe the conclusions and implications

Organize and group related ideas together

Identify the references that pertain to each key point

Medical School for International Health

TEACHING UNIT: Bacteriology Lab 1

General Techniques in Bacteriology Lab.

Collection and transport of clinical specimens.

Collection and transport of clinical specimens

Cultivation and Isolation of bacteria

Preparation of pure cultures by streaking plates.

Streaking for enumeration of microorganisms

Specialized Culture Media and Culture Conditions

Selenite broth is a peptone base broth containing sodium selenite

MacConkey agar selects against Gram-positive organisms due to

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

Sheep Blood agar allow differentiation of hemolytic reactions of

Blood agar is supportive for many microorganisms and allows

Optical Methods for diagnosis

Preliminary identification of colonies growing on solid media

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

Testing antimicrobial susceptibility of aerobic bacteria

September 24th, 15:00-17:00 hours.

Escherichia coli; Bacillus subtilis; Salmonella;

A mixture of two microorganisms.

Streaking for purified colonies:

Streaking for enumeration of microorganisms in urine:

Testing culturing media and conditions:

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

Streak Haemophilus on Chocolate agar plate using scheme 2.

Testing antimicrobial susceptibility

Using a dispenser, apply antibiotic disks.

Invert plates and incubate overnight at 37C.

Inoculating urine samples using Dipsticks

Different Agar media

Bacterial resistance examination

Examine plates for purified colonies, and evaluate your performance.

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

Compare antimicrobial susceptibility of all microorganisms.

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

Perform Ziehl-Neilsen staining of pre-fixed mycobacterium.

Summarize the morphological characteristics of the microorganisms examined in this

Results table for laboratory #1

Appendix 1: Culture media

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

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

* Adjusted and/or supplemented as required to meet performance criteria. Final pH 7.10.2

* Adjusted and/or supplemented as required to meet performance criteria. Final pH 7.30.2

Appendix 2: Bacterial Cultivation

Preparation of evenly coated plates:

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

Medical School for International Health

TEACHING UNIT: Bacteriology Lab 2

Gram-Positive Cocci : Staphylococci and Streptococci

Performing laboratory tests leading to characterization and diagnosis of

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

On nutrient agar (NA):