Microbiology Unit - Bacteria Lesson VSuggested reading: Hendrix and Sirois pp. 128-141, McCurnin pp.
240-259, Quinn and Markey pp. 8-13
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. .
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.
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.
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.
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.
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.
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.
Colony characteristics and differential reactions on the plates are recorded.
Subculture colonies to develop and maintain a pure culture.
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.
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 ?
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.
Click here for an example of the oxidase test: http://medic.med.uth.tmc.edu/path/oxidase.htm
What if it's a large Gram positive bacilli?
What if it's a small Gram positive bacilli?
Then other tests are utilized to differentiate further.
Here is a summary flow chart for differentiations of small Gram positive bacilli
What if it's a Gram negative bacilli?
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.
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
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.
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.
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.
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.
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.
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.
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.
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.
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)
Click here to view a Mycobacterium slide
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.
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:
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:
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.