Microbiology Unit - Bacteria Lesson V

Suggested reading: Hendrix and Sirois pp. 128-141, McCurnin pp. 240-259, Quinn and Markey pp. 8-13  

Key Word List | Testing and Identification of Bacteria | Writing Assignment

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Key word list

  • Culture and sensitivity
  • direct inoculation
  • primary plates
  • enrichment culturing
  • thioglycollate
  • Blood agar
  • MacConkey agar
  • isolation streak
  • isolate
  • swarmer
  • catalase test
  • coagulase test
  • oxidase test
  • TSI reaction
  • butt and slant
  • antibiotic sensitivity testing
  • Bauer Kirby
  • MIC
  • serotyping
  • DNA typing
  • Profiles
  • Bergey's Manual

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Guidelines for bacterial identification

The specimen has been collected and now transported to the lab for culture, identification  of the bacteria, and antibiotic testing. Some practices may have an in-house microbiology  lab, while others may send specimens to an outside lab. In any case, the lab technician  must first culture the specimen, separate the bacterial populations into pure cultures, and  perform tests to identify the bacteria.

There are countless tests and media for growing bacteria and so many different kinds of  bacteria, it can be overwhelming at first where to start and how to figure out what bacteria  are in the specimen. All you have to do is glance at Table 8-8 in McCurnin and Tables 4-2 and 4-3 in Hendrix (or Hendrix and Sirois) as well as all the biochemical reaction tables in both texts to appreciate the  large numbers of pathogens and tests there are. But the basic approach to identifying the  bacteria species in a specimen is to follow a predetermined step-by-step method of rule outs until you narrow the possibilities down to one species of bacteria. The protocols used  in the identification method will vary with the type of specimen, the animal the specimen  is from, and the suspect pathogens in the differential diagnoses. . 

ruleoutmethod.gif (6697 bytes) Sample schematic of a rule out approach to identify a hypothetical bacterium that is a Gram positive cocci, testing C and Z positive.

With so many choices and scenarios, how does one know what to do?

All laboratories have specific protocols for the technicians to follow that are determined  by the laboratory director who also supervises the technicians and the interpretations of  results. Years of research have established for us what methods and tests usually work  best for each situation. The lab protocols are very similar from lab to lab, but will differ  some according to the training and preferences of the lab director. 

All laboratories will have a lab manual of standard operating procedures that guides the  technician in the choice of media and tests depending on the specimen submitted and any  other relevant clinical information supplied to the lab. Flow charts are provided for each  protocol to arrive at the identification of the bacteria species.  

How do we know which protocol to use?

We use the information about the specimen when it arrives in the lab.  We know from  years of research and retrospective case studies that certain pathogenic bacteria infect  certain animals, certain tissues in those animals, and cause certain diseases. There will  be different lab protocols recommended for particular specimens, animal species, and  clinical signs. For example, look at Table 8-1 in McCurnin and Bassert on pages 238-239. You  will see a listing of the type of infection and then a listing of the most likely pathogens  for each species of animal that the specimen is from. This information directs the type of media and tests that are requested and chosen for culturing the specimen: 

Example 1 Table 8-1 McCurnin and Bassert

Let's look at a case of skin wounds and abscesses in the cat. There are three aerobes listed  as the most likely candidates: Pasteurella multocida, Streptococcus, and  Staphylococcus. These three organisms are readily differentiated by routine aerobic  culture techniques. However, anaerobes are also listed as common inhabitants of cat  abscesses. Therefore, in addition to performing a routine aerobic culture, the lab  manual would advise us to also use a medium that would support anaerobes, such as  thioglycollate. Otherwise, we risk missing a lot of important information about the  infection.  

Example 2 Table 8-1 McCurnin

Let's look at a case example of upper respiratory infection in a steer. For this case, a nasal swab is  submitted, but it is noted on the form that the steer may be developing neurologic signs.  For upper respiratory infections in ruminants, the likely candidates are Hemophilus somnus, A. pyogenes, and Fusobacterium. For central nervous system infections of  ruminants, the likely candidates listed are Hemophilus somnus, Listeria, E. coli, and Pasteurella multocida. With this information, we see that H. somnus is a very likely  candidate. And because H. somnus is a fastidious organism, the lab protocol for bovine  upper respiratory specimens will direct you to use a highly enriched media like chocolate  agar as well as routine media on the upper respiratory specimen from the steer. 

Example 3 Table 8-1 McCurnin

Now look at the case of an upper respiratory infection from a dog from which a nasal  swab has been submitted. This same type of specimen from a dog would be handled  differently than that of the steer. The most common organism found in the dog is Bordetella bronchiseptica, which will grow on routine non-enriched media. For this  reason, the lab manual would in this case direct you to use routine techniques on the  upper respiratory specimen from the dog.

From these examples, you can see culture protocols will not be the same for every  specimen. For that reason a lab manual will have many protocols developed and you  would choose the protocol that is designed for a particular specimen.

Now that we have the protocol, how do we perform and interpret all these tests?

There are many tests available, and new tests are constantly being developed. The tests  detect the various metabolic activities of an organism, which could include enzyme or  toxin production, utilization of nutrients, and physical traits. The protocol that is chosen  for the specimen will outline in flow chart fashion what tests to perform and in what order  to perform them. The tests are usually commercially provided, and interpretations of the  test results are either packaged with the test, in a lab reference manual, or textbook.

Generally, many of the tests use a color change to determine a positive or negative result.  Reagents are used in the media that will change from one color to another if a certain  nutrient is utilized or if a certain substance is produced. For example, organisms that  ferment carbohydrates produce acid waste products which then lower the pH of the  medium. A pH indicator in the medium will turn a certain color when the pH reaches a  certain acidic level. If the medium only contains one sugar, like lactose, then the only  reason the medium would turn color is if the organism ferments lactose. So if there is the  designated color change, then the organism is said to be lactose positive, because it  ferments lactose. If there is no color change, then the organisms is said to be lactose  negative, because it does not ferment lactose. 

Besides biochemical reactions, there are other tests available to determine the presence of  certain organisms. Evaluation of stained smears is often helpful in the identification of the  organism before it has even been sent to the lab for culture. Stains such as the Gram stain,  acid fast stain, or fluorescent antibody stains are used on exudates and other specimens.  Serotyping is used to identify a specific characteristic antigen of an organism by using an  antibody-antigen reaction test on the bacterial culture to detect the tell-tale antigen.  Sometimes acute and convalescent serum antibody titers of the patient are used to verify the  presence of a pathogen that is not easily cultured.  More recently, the detection of DNA  and RNA sequences unique to an organism can be performed on even small samples, thus  identifying the presence of the organism in the specimen.

Once a battery of tests is completed, we then have developed a profile of the organism as  to its various traits, both positive and negative results.  We then match that profile up with a known organism and the identification is accomplished.  

Overview of routine aerobic culture protocol

Let's go through a typical aerobic culture protocol. Please note that the pathogens listed  here are not a complete list, but are just intended to highlight a few well known ones so  you can see how the flow charts work with the classification of organisms. 

For midterm exam purposes, you should know about the tests specifically discussed below and  know how to work through a flow chart, but please do not memorize these flow charts  and do not memorize all those tables in the textbooks. 

  • Step 1 - Determine the protocol

The specimen that arrives at the lab will be either a swab, liquid, or tissue sample.  Accompanying the specimen will be information on the patient species, the source or site of the specimen, and clinical signs, as well as differential diagnoses. Ideally, a sample of the specimen has already been Gram stained at the time of collection to help insure that the right organisms are being isolated and identified in the lab. At this point, you  will check the lab manual listing for the specimen to determine which protocol is the  most appropriate.  In this scenario, routine culture media is recommended.

You will record all your work, daily progress notes and results on the lab microbiology  worksheet form for each specimen and each isolate. The data you record documents your  findings should there be any questions later.

  • Step 2- Grow the bacteria   

The specimen is used to directly inoculate blood agar and MacConkey agar plates in an  isolation streak method. The specimen may be inoculated in the first section of the agar  plates, then completed using a loop as previously described. If there is enough specimen volume, a Gram stain may be performed on a sample of the specimen the first day.

Specimenprimaryplate.gif (2693 bytes)

For urine, a colony count plate is also set up.

The specimen is also then inoculated into an enrichment broth which encourages growth  and enhances the bacterial numbers of a small bacterial sample; any additional growth in  the broth can be used later for subculturing if needed as back up, if growth on the plates fail. It is ideal if the broth will support anaerobes.   

Incubate at 37 degrees C  for 12-24 hours.

  • Step 3- Isolate   

If no growth on anything: inoculate enriched media for fastidious organisms and  reincubate all plates and broth, checking daily.

If there is no growth on the primary plates, but there is growth in the broth, then perform  an isolation streak of the enrichment broth culture on blood agar and MacConkey agar.  Incubate.

  • Step 4- Establish pure cultures and begin testing   

Once growth is established on plates, the colony types and relative numbers of colony  types are observed. 

Colonies are chosen for subculturing and Gram staining.

primarybloodagar.gif (3648 bytes)

Colony characteristics and differential reactions on the plates are recorded. 

Some organisms produce a characteristic odor. For example, Proteus sp. produces  an odor that some people describe as stinky or putrid. Pseudomonas sp. produces  a pungent fruity grape odor. I wish I could do a "scratch and sniff" attachment  here for you, but I can't. Don't worry, you won't miss it when you smell it,  believe me!

If pigment is produced- this is also a differentiating characteristic. For example,  some species of Pseudomonas will produce a  blue-green pigment. This pigment will readily diffuse into solid media so you will  notice the change in color easily on nutrient agar, which is normally only a tan  color. 

Click here to see an example of pigment production:

http://textbookofbacteriology.net/pseudomonas.html

 

For blood agar we also note hemolysis patterns: Alpha, beta, double zone, or no  hemolysis.

See this website for examples of blood agar hemolysis patterns.
 

http://gold.aecom.yu.edu/id/micro/hemolysis.htm

http://web.indstate.edu/thcme/micro/hemolys.html

For MacConkey agar we note if there is no growth or growth. If there is growth,  the organism is likely in the enteric group. We note if the colony is a lactose  fermenter (pink growth reported as lactose positive) or does not ferment lactose  (lactose negative).

See this website for examples of MacConkey agar growth.

http://www.austincc.edu/microbugz/html/macconkey_agar.html

Subculture colonies to develop and maintain a pure culture.

subculturestep.gif (9933 bytes)

Sometimes organisms cannot be separated because there may be an extremely motile  organism present that is capable of even moving over solid media and contaminating  separate colonies. When this happens, you will see a film of growth overlaying the  isolated colonies-- an organism that exhibits this strong motility is referred to as a  "swarmer". Proteus sp. are notorious for swarming. Special media must then be used to  inhibit the swarming activity so that the organisms may be isolated into pure cultures.

  • Step 5- Testing and identification 

Gram stain reaction and morphology now determines the next set of identification  procedures. Using pure cultures, perform tests sequentially to work through a flow chart  to identify the organism and set up an antibiotic sensitivity plate. 

The very first identification test is to perform a Gram stain procedure. The morphology and cellular arrangements as well as the Gram reaction are determined with this simple test. Once we know whether the organism is Gram positive or Gram negative, and whether it is a coccus or bacillus, we start our journey down the flow charts.

Let's review a few examples of bacteria in the aerobic group.

What if it's a Gram positive cocci ? 

The two main tests we use for this group initially are the catalase and coagulase  tests. Then other specialized tests differentiate the species of bacteria further

Catalase test- catalase is an enzyme that will form oxygen bubbles from hydrogen peroxide. 

catalasetest.gif (3822 bytes)

Click here for a website that shows an example of a catalase test  http://medic.med.uth.tmc.edu/path/catalase.htm 

Coagulase test- coagulase is an enzyme that will clot plasma.

coagulasetest.gif (3635 bytes)

Click here for a website that shows an example of a coagulase test http://medic.med.uth.tmc.edu/path/coag.htm 

1. First perform the Catalase test:

If Catalase negative: Streptococcus group

Streptococcus species tend to form pairs or chains of cocci. Further testing of the catalase negative organisms will differentiate more specifically what organism is present. Examples of well known veterinary pathogens in this group include

Streptococcus agalactiae (mastitis in cows)

Streptococcus equi  ("strangles" in horses) 

If Catalase positive: Staphyloccocus group

Staphylococcus species then to form clusters of cocci. Further testing of the catalase positive organisms will differentiate more specifically what organism is present:

2. Now perform Coagulase test on the Catalase positives:

If Coagulase positive-- usually a pathogen! Further testing of the catalase positive/ coagulase positive organisms will differentiate more specifically what organism is present. Here are some example of what pathogens might be in this group: 

Staphylococcus aureus  (skin infections)

Staphylococcus intermedius 
famous skin pathogens (pyoderma) will infect many sites 

Staphylococcus hyicus (greasy pig disease) 

For photos of typical Staphylococcus aureus bacteria:

http://www.meddean.luc.edu/lumen/DeptWebs/
microbio/med/gram/slides/slide3.htm

If Coagulase negative: other Staphylococcus species

Here is a summary flow chart of the differentiation of some Gram positive cocci

flowchartGposcocci2.gif (6075 bytes)

What if it's a Gram negative cocci?

There are not very many veterinary pathogens in this group. Better double check  your controls on the Gram stain to make sure on this one!  The test used to identify the pathogen in this group are catalase and oxidase test.

The oxidase test is a test used to detect the presence of cytochrome oxidase, an enzyme of  a specific energy pathway. A positive test turns the reagent purple.

oxidasetest.gif (3117 bytes)

Click here for an example of the oxidase test: http://medic.med.uth.tmc.edu/path/oxidase.htm 

If we have a Catalase positive and Oxidase Positive, we might have: 

Moraxella bovis 

Sometimes listed as a small Gram negative coccobacillary  organism or a small Gram negative bacillus, may appear to be a  diplococcus. It is the causative agent of  contagious keratoconjunctivitis in cattle which sometimes leads to blindness.

What if it's a large Gram positive bacilli?

Then first , we check the Hemolytic reaction on Blood agar. If we have hemolysis present, then we have the group: 

Bacillus species, some known to cause food borne illness

If there is a Nonhemolytic reaction on blood agar we might have: 

Bacillus anthracis  (yikes!!!) causative agent of anthrax- exotoxin and endospores produced, often sudden death is the only sign in the patient


PHIL_1064_lores.jpg (95995 bytes) image courtesy of CDC/Dr. William A. Clark

Here is a summary flow chart for differentiations of large Gram positive bacilli

flowchartlargeGposbacilli.gif (3319 bytes)

What if it's a small Gram positive bacilli?

The catalase test is the first test used in differentiation of this group.

If Catalase negative some likely candidates could include 

Erysipelothrix rhusopathiae (diamond skin disease in pigs) 
Actinomyces pyogenes (various)

If Catalase positive some possibilities include

Listeria monocytogenes (meningitis) 
Corynebacterium sp.(various skin and respiratory infections)

Then other tests are utilized to differentiate further.

Here is a summary flow chart for differentiations of small Gram positive bacilli

flowchartsmallgramposbacill.gif (5593 bytes)

What if it's a Gram negative bacilli?

The initial test for differentiation of this group is the oxidase test

If Oxidase test positive

Common pathogens in this group include but are not limited to

Pasteurella multocida (pathogen and often present in the oral flora of  cats-beware of cat bite wounds!)

Pasteurella hemolytica (respiratory infections "shipping fever" in cattle)

Hemophilus somnus- (respiratory and central nervous system  infections "shipping fever")

Bordetella bronchiseptica ("kennel cough" in dogs) 

Moraxella bovis (oops! there it is again!sometimes it looks like a short stumpy Gram negative bacillus)- contagious conjunctivitis in  cattle

Brucella sp. (reproductive system- brucellosis, Bang's disease,  undulant fever)

Pseudomonas (wounds, ear infections- may be considered to be an opportunistic infection, often antibiotic resistant)

If Oxidase negative

various Enterobacteriaceae (enterics)- this is a huge group!

Common pathogens include but are not limited to

Escherichia coli  (pathogenic strains of E. coli) 

Salmonella  various species (gastroenteritis) 

Yersinia  pestis (bubonic plague)  

For Enterobacteriaceae, then perform TSI slant inoculation for presumptive identification and incubate. The TSI slant performs several reactions in the same tube.

Results are reported separately for the bottom or the "butt" of the tube and for the  top or the "slant" of the tube. If there is color change to yellow it is reported as "acid" or "A". If there is no color change it is reported as "alkaline" or  "K". The slant is reported first then the butt, using the letters "A or "K". So if both  are acid, the result is reported as A/A. Gas is sometimes produced and displaces the  agar. If hydrogen sulfide is produced, then there is a black color produced.

TSIdiagram2.gif (12744 bytes)

Click here for more explanation and examples of TSI reactions. http://medic.med.uth.tmc.edu/path/triple.htm 

Here is a summary flow chart for differentiation of Gram negative bacilli

flowchartgramnegbacilli.gif (5942 bytes)

Commercial I.D systems

There are commercial identification systems that can simplify and streamline the  testing and identification process. A popular example is the API system. API set ups  are comprised of a small plastic tray containing miniature tubes with various reagents.  An entire battery of tests may be performed in the tray that normally would take an  entire rack of test tubes and use much more reagent and media.  With this system a  code number is generated based on the combination of positive and negative results;  positive results give points, while negative results give a zero. Using statistics, the  code number is then matched up to the most likely organism with the same number.  These test kits are often used to confirm the presumptive identification from the flow  chart.  

APIset1.jpg (27314 bytes)     APIset3.jpg (24438 bytes) Here is an example of an API system tray set up.

api_form2.jpg (198320 bytes) Here is an example of an API code number report form.

Antibiotic Sensitivity testing

When identification test are set up, antibiotic sensitivity testing is also set up. There are two main methods used for testing antibiotics against an organism.

Minimum inhibitory concentrations (MIC) of antibiotics are determined by making serial dilutions of each antibiotic in nutrient media and culturing the organisms in these solutions. The lowest concentration of antibiotic at which growth is inhibited is reported as the MIC. This gives the clinician an idea of what dose of antibiotic to use.  The test is somewhat laborious and is not routinely used in veterinary medicine.

The agar diffusion method of antibiotic sensitivity testing is more often used because it is  easy to do and gives information about several antibiotics with one test. In the method  known as the Bauer-Kirby technique, a Mueller-Hinton agar plate is inoculated with the  bacteria. The inoculum is evenly distributed over the entire plate, ultimately creating a  film of growth known as a "lawn". At the time of inoculation, small paper discs, each  containing an antibiotic, are placed onto the plate also. During incubation, the antibiotic  diffuses out into the agar a certain distance from each disk. If the bacteria are susceptible   to the antibiotic, they will not grow around the disc where the antibiotic is present in the  media. The inhibition of growth can be seen as a clear zone around the disk. If the  bacteria are resistant, they will grow right up to the edge of the disk. If the zone of  inhibition is small, there is some inhibition of growth, but not enough to be considered  "susceptible"; this is called "intermediate". The sensitive zone measurements are different for each antibiotic and are standardized by the manufacturer using Mueller  Hinton agar. Generally, this test gives the clinician an idea of which antibiotic may work  for the particular case from which the organism came.    

Click here for examples of antibiotic sensitivity testing set ups.

http://gold.aecom.yu.edu/id/micro/kirbybauer.htm

http://www.biotopics.co.uk/microbes/sendsk.html

Regardless of the methodology used, the bacteria tested against the antibiotics must be a  pure culture. If not, then the overlap of antibiotic resistance of two or more organisms  will not reveal an appropriate combination of antibiotics. 

sens.gif (8560 bytes) Diagram showing how antibiotic sensitivity results are affected by the use of mixed  cultures, giving erroneous results.

  • Step 6- Compile and verify results

Record the identification and antibiotic sensitivity results for each organism isolated.  Verify that your findings agree with what is known about the organism such as Gram  stain and morphology, cellular arrangements, colony characteristics, broth growth  patterns, and differentiating tests. 

Why do we need to verify our findings if the results are reliable and follow the flow  chart?

The annoying and tricky part of verifying your findings is that biochemical reactions for  organisms are actually reported on a percentage basis in research rather than just either  positive or negative. There can be some variation within a species for some traits. For  example, if research studies show that an organism is usually positive for lactose  fermentation about 98% of the time, then that means that 2% of the time, there could be a  member of the species that is lactose negative. The flow charts in lab manuals are based  on the most common biochemical reactions found for a species. Therefore, it is possible  that if you happen to isolate one of the unusual deviants, it could affect the path of the  flow chart and lead you to an incorrect identification. So when interpreting results, you  must play detective and keep in mind that there can be variations for some profiles of an  organism. 

Here is an example of the common biochemical reactions of 2 organisms - E. coli and  Yersinia pestis. They are very similar and could overlap in identification, but they are  very different organisms and cause very different diseases. Yersinia pestis is the causative  agent of bubonic plague, E. coli is not. The numbers in the table indicate the % of the organism population that has been found to be positive.

 

Yersinia pestis

E. coli strain 2

Citrate

0

0

Hydrogen sulfide

0

1

Urease

0

1

Glucose

99

99

Mannose

99

90

Sorbitol

70

42

Oxidase

0

0

Indole

0

50

Gelatin

0

0

In any case, you would check in a reference bacteria manual such as Bergey's Manual for  such information and make sure your final results make sense. Then report your findings.  On average, this entire process of bacterial isolation, testing, and identification takes 3 to  5 days.

What about filamentous bacteria?

Filamentous bacteria are bacteria that are pleomorphic, but generally appear as bacilli that form branching strands as well as clumping in palisading or Chinese letter formations. Some form coccoid elements as well. Trying to decide what the morphology is can drive you crazy! Many of the organisms are presumptively identified by a specimen smear showing the typical morphology of the species. Some are anaerobic and difficult to culture. 

Actinomyces sp. (oral infections, lumpy jaw in cattle)

The exudate will often have yellow granules: “sulfur granules”. The organism may be demonstrated by crushing the granule on a slide and Gram stain.           

Dermatophilus congolensis

Dermatitis in horses, ruminants- dermatophilosis, streptothricosis, “rain rot”, “rain scald”, “creeping crud”, “lumpy wool”

Horse owners in Virginia sooner or later experience this on their horses. It can be zoonotic so wear gloves when cleaning those scabs off your horse. 

scabs.jpg (104092 bytes) In horses, the lesions form scabs that when exfoliated also take the hair with them, resulting in the typical “paintbrush scab”

Branching filaments and clumps of paired coccoid elements are seen by soaking the scabs in water or saline and then gently preparing a smear of the moistened material. Or a swab of exudate from the lesion may be adequate. A stain like Diff Quik or new methylene blue is adequate to view the organism. 


PHIL_2986_lores.jpg (33385 bytes) The Dermatophilus congolensis organism, image courtesy of CDC

Nocardia asteroides- also acid fast as well as a branching filamentous bacillus

Nocardia causes abscesses in dogs- the organism can be demonstrated by staining the exudate which may also contain whitish granules.

  Nocardia asteroides courtesy of CDC Dr. Lucille Georg

What about acid fast bacteria? 

The most notable acid fast group are the Mycobacterium species.

You will not likely culture these organisms in your lab routinely as they can be fastidious and grow very slowly, sometimes taking several weeks, and you will have discarded your culture as “no growth” by then. However, they can be viewed in stained smears using the acid fast technique and will appear as clumped acid fast rods.

Purified substances from these organisms are often used to test for hypersensitivity in the host to check for exposure to the organism. One of the most common tests used in veterinary medicine is the tuberculin test for tuberculosis.

Here are some examples of pathogens in the Mycobacterium group:

M. bovis (tuberculosis)
M. avian (avian tuberculosis)
M. paratuberculosis (Johne’s Disease)
M. leprae (leprosy) 

Click here to view a Mycobacterium slide
http://www-medlib.med.utah.edu/WebPath/INFEHTML/INFEC037.html

What about spirochetes or curved bacteria?

Spirochetes are often fastidious and difficult to culture. Although most are Gram negative, sometimes they are very thin and difficult to demonstrate on routine stained smears. Therefore, many times other tests are used to detect the organisms, such as special microscopy techniques of a specimen, special stains, or serology.

negative_sprio.jpg (18561 bytes) Negative stain preparation containing spirochete and curved bacteria examples.

Here are some notable pathogens:
Leptospira sp- urinary tract infections and abortions
Helicobacter mustelidae- gastric ulcers in ferrets
Borrelia species – the most famous member causes Lyme’s disease             Campylobacter species- intestinal and reproductive tract infections

special stain- Campylobacter jejuni courtesy of Dr. Robert Weaver. many technicians remember Campylobacter as looking like seagulls flying in the distance.

  Campylobacter fetus courtesy of CDC

What about anaerobic culture?

Anaerobic cultures follow the same basic concepts of identification except that  during culturing techniques, the organisms are protected from oxygen. The organisms  are incubated in an oxygen depleted atmosphere which is acquired by the use of  devices and chambers that displace room air and prevent it from reentering the incubation chamber.

A simple technique used to decrease the oxygen and increase carbon dioxide is the candle jar. Cultures are placed in a jar with a lit candle and the jar is sealed. As the candle burns out, the oxygen is depleted to a lower than normal level and the carbon dioxide increased. However, this technique does not create the strictly anaerobic environment required by some organisms.  

For those organisms that require a strict anaerobic environment, anaerobic chambers that allow the operator to work through a glove box have been devised. Click here for more explanation and pictures of anaerobic culturing techniques using an anaerobic chamber:

http://www.shellab.com/bactron.html

Since proper anaerobic specimen collection and culture can be difficult, often we use a stained smear of the specimen to make an educated guess as to what group of bacteria might be causing an infection in our patient. It is important for you to be able to recognize certain staining patterns and properly describe the organisms you see on a specimen smear.

Anaerobic pathogens include but are not limited to:

Anaerobic Gram positive endospore formers 

The Clostridium group contain bacilli that form endospores and exotoxins. Many notable pathogens-- It’s a scary group! They are often presumptively identified by the presence of spore-forming activity on cytology. 

C. tetani (tetanus)
C. botulinum (botulism)
C. difficile (intestinal sites)
C. novyi  (black disease)
C. chauvoei (black leg)
C. septicum (malignant edema)
C. perfringens (enterotoxemia, overeating disease)
And many others! 

  Clostridium difficile courtesy of CDC Dr. Gilda Jones. Many technicians remember this organism as resembling a safety pin or paperclip because of the endospore position and staining characteristic

Clostridium botulinum courtesy of CDC Dr. George Lombard. Technicians describe the terminal endospores as resembling clubs or tennis rackets.

Anaerobic Gram negative                                                                

Bacteriodes sp.- associated with periodontal disease and foot rot (some  species renamed Porphyromonas )
Fusobacterium necrophorum- associated with other organisms in foot rot infections of large animals, and "thrush" in horses 

Fusobacterium necrophorum courtesy of CDC Dr. VR Dowell, Jr.

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Lesson 5 - Writing Assignment:    

Learning questions for online lesson: answers are to be completed in a word document downloadable from the college Blackboard site and uploaded into the assignment drop box. All SECTION I assignments are due before taking the first exam.

1. If there is growth in thioglycollate broth, but no growth on the plates, what does that  mean?

2. When we have numerous colonies on the first set of isolation plates, how do we  know which colony to pick for identification and the development of a pure culture?

3. If a specimen reveals that there are 2 organisms present, why not just test both on the  same plate for antibiotic sensitivity to save on time and money?

4. Why not just do an antibiotic sensitivity test right away off the specimen? After all the  bottom line is to find out what drug to use. Why should we care what organism is  there?

5. For how long do we incubate plates that have no growth before we give up and report  no growth?

6. You may have noticed that on some of the differentiation charts, the results are  reported as +, -, or V. What is "V"?

7. What important aerobic large Gram positive bacillus pathogen is usually non-hemolytic?

8. What organism (presented in these notes) produces a pigment that could make exudate appear as a bright yellowish green color?

9. What is the significance of methicillin-resistant  and vancomycin-resistant  organisms? If you can't find the info in the reading, ask your mentor, or go to http://www.fda.gov/fdac/features/795_antibio.html  and http://www.mayoclinic.com/health/mrsa/DS00735

10.What is a coliform?

11. Describe the 3 main types of hemolysis patterns on blood agar.

12. Describe the difference between a lactose positive and a lactose negative organism growing on MacConkey agar.

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