Do they live as single cells or in a multi-cellular arrangement? Many other traits of large organisms can be cataloged and used for identification. Microorganisms are small, and frankly there are not many morphological traits than can be tracked.
However, microbes are extremely versatile and varied in their metabolic traits. By growing them in various medium [6] to test their metabolic capabilities, it is possible to identify microbes. For example, most Salmonella strains cannot metabolize lactose, while E. By testing enough traits it is possible to identify an isolate to the species level and beyond. Typical tests Many biochemical and physiological tests have been developed to identify bacteria [7].
The tests you will observe in this experiment fermentation [8] broths, starch hydrolysis, colony morphology, catalase test, motility agar and indole from tryptophan are useful in microbial identification. Gram stain The Gram stain is a primary method for differentiating microbes. The reaction of any bacteria to the stain depends upon it cell [9] wall structure. The Gram stain was covered in detail in Section [10] Figure Gram reactions of strains The Gram stain reactions of common microbes.
If the test sugar is fermented, acid is usually produced. This drop in pH turns the brom cresol purple from blue to yellow.
Also, the presence of a Durham tube, allows the detection of gas H2 production from the fermentation. Figure Reactions in fermentation broth Example reactions of typical strains in fermentation broth. If a microbe does not ferment the test sugar, the indicator dye remains purple and no gas is produced left. If fermentation does take place, acid is most often produced, lowering the pH and changing the color of the broth to yellow middle.
Some microbes produce hydrogen gas during fermentation and this will be trapped inside the inverted tube, called a Durham tube, and is observed as a bubble right. Starch Hydrolysis The starch agar plate is used to test for extracellular starch hydrolysis.
Starch is a high molecular weight polysaccharide, too large to be transported inside the cell without breaking it down into smaller units first. The ability to hydrolyze starch depends upon the production and secretion of several amylases that degrade the polymer.
The breakdown of starch is detected after incubation by flooding the plate with iodine. Iodine complexes with intact starch to form a blue color. If the microbe is able to degrade the starch, none is available for reaction, and a zone of clearing will be seen around the colony. Microbes capable of utilizing the starch will secrete amylases, which hydrolyze the starch.
Starch breakdown is detected by flooding the plate with iodine, which forms a purple colored complex. Test microbes incapable of degrading the starch will be completely surrounded by a zone of purple. Colony Morphology and Catalase Test HIA is used as an all-purpose medium, that will allow the examination of colony morphology and catalase activity. The correct method for describing colony morphology is described in Figure [13].
As mentioned in Experiment 3, some oxygen by-products, created during metabolism [14], are toxic to cells and special enzymes have evolved to detoxify those compounds. One of these enzymes, catalase, is responsible for the splitting of hydrogen peroxide H2O2 into oxygen and water. It is easy to test for this enzyme in bacteria. The oxygen is detected as a steady evolution of gas bubbles from the culture.
Figure [15] shows a movie of the catalase reaction. The common descriptive names for colonies [16] are described in Figure [17] and examples of colony morphology are shown in Figure [18]. Individual cells or a few identical cells will continue to grow and divide and form discrete units called colonies, the morphology of which is characteristic of that microbial species.
An accurate description of an isolated colony can greatly aid in the identification of the microorganism. Microbiologists have developed standard words with specific meanings to describe colony form, elevation and margin. Scientists use this jargon to help accurately communicate their observations.
This is a common theme in all disciplines of science. Imagine what would happen if everyone used different terminologies? There would be no effective communication between scientist. Figure Colony morphology Examples of varous colony morphologies. The appearance of colonies on a plate is species specific and can be very helpful in identifying isolates. Tryptophan Degradation to Indole Tryptone broth contains a high concentration of the amino acid tryptophan.
Some microorganisms are able to degrade tryptophan to indole and this ability is a useful tool for differentiating microbes. The test is performed by adding Kovac's reagent to a broth culture, which results in a red ring on the top of the broth if indole is present. Identifying of microorganisms through these types of biochemical tests has been a routine practice for many decades and the tests necessary to identify certain species of groups of related species have become standardized.
To make these test more convenient, manufacturers have developed miniaturized versions that decrease the material cost and make inoculation [20] of the tests much more convenient. However, the tests still need to be incubated to the prescribed period of time before being read. Usually hours. One example of this type of test is shown in Figure [21], the API 20 strip.
The types of biochemical tests we have explored here can be miniaturized. The API strip is one example of this type of test. Twenty tests are performed on this strip by a simple procedure, saving time and money. The first test is for the presence of the enzyme?
The next three reactions in order, arginine, lysine and ornithine test for amino acid decarboxylation. Decarboxylation is shown by an alkaline reaction red color of the particular pH indicator used.
A positive reaction for tryptophan deaminase TDA gives a deep brown color with the addition of ferric chloride; positive results for this test correlate with positive phenylalanine and lysine deaminase reactions which are characteristic of Proteus, Morganella and Providencia.
The last nine tests are for carbohydrate fermentation. The carbohydrates tests are glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, melibiose, amygdalin and arabinose.
Fermentation is shown by an acid reaction yellow color of indicator. In Experiment 7 the student will first become familiar with several diagnostic tests and the reactions of known organisms. You will then be given a virtual unknown and will apply these tests in the hope of identifying your microbe. By comparing the results of your tests to a virtual unknown you will identify your microbe.
To simulate real life, the reactions of microbes in medium can sometime be ambiguous, and we will simulate this by sometimes having unclear or incorrect results. You have been warned. Read through it to get an idea of the steps necessary to perform the tests. Each student will be responsible for observing each test on every organism!
It is usually most convenient to record data on the characteristics of microorganisms in tabular form. Perform a Gram [24] stain on each of the pure cultures. Note the cell [25] morphology and Gram reaction and be sure to examine the Gram stains prepared by other members in your group. Inoculate both of the fermentation broths with the cultures. Avoid shaking the tubes since this may trap an air bubble in the Durham tube, confusing the results.
Streak a single line of each culture onto the middle of a plate of starch agar. Do not streak for isolated colonies [26]. Streak each culture onto a plate of HIA for isolated colonies. Inoculate each culture into a tube of tryptone broth to test for the production of indole from tryptophan.
Tryptone is a peptone that contains high concentrations of tryptophan. Nitrate reduction [27]. Inoculate a tube of Nitrate Broth. Observe the fermentation broths. First, look for turbidity [29] in the tube, record this as growth.
Second, examine the color of the medium. The original color was purple alkaline , the fermentation of the sugar to acid causes the medium to turn yellow acidic. Record whether your organism ferments the sugar to acid. Finally look for a bubble in the Durham tube; if present it signifies the production of gas. Observe the streak on the starch agar plate.
Then gently flood an area around growth with Gram's iodine. Allow the plate to react for about 2 min. Starch will react with the iodine to form a blue complex. If the starch has been hydrolyzed by the extracellular enzyme amylase, a clear zone will be seen around the streak.
Record this as a positive result. If the blue color runs all the way to the edge of the growth record this as a negative result. Observe the colonies on HIA and record your results. Are all the colonies of the same general type? If they are not, what does this mean? Describe the pigmentation, opacity, form, elevation and margin of the colonies. Refer to Figure [30] for the correct terms to use. Place the lid back on the plate and watch for the production of bubbles.
The appearance of a constant evolution signifies the presence of the catalase enzyme. Carefully layer half a dropperful of Kovac's reagent onto the surface of the tryptone broth cultures and let sit for a few minutes. The development of a red ring at the broth reagent interface is a positive test for indole production. Note that only one of the known cultures is expected to give a positive test for indole.
Nitrate reduction. To observe for the reduction of nitrate in the Nitrate Broth all the way to nitrogen gas i. To test for the reduction of nitrate only to nitrite, add a dropperful of each of the two nitrite reagents - sulfanilamide and N- 1-naphthyl -ethylenediamine - to each of the tubes. Mix well. Any appearance of a red color which may or may not persist indicates the presence of nitrite.
If no red color was seen, add a few more drops of each reagent, mix well, and observe again. If no indication of gas or nitrite was seen, the nitrate may not have been reduced, or it was reduced to a product for which we are not testing. Mix the tube well. If nitrate is present, it will be reduced by the zinc to nitrite which will react with the reagents already added, forming a pink or red color. Disregard any gas generated with the addition of the zinc.
Do not record for the organism any reaction you see only upon addition of the zinc. Because of variation in incubation time, size of inoculum and variable skill in the experimenter, results for each species may be slightly different.
With that caution, familiarize yourself with these. Pay special attention to the colony morphology, as that can be very distinctive for each species. Figure Gram stains of 9 isolates Culture Reaction Description This microbe appears as gram-negative Escherichia coli short rods.
Older cultures more than 24 hours may appear as cocci. This microbe may appears as gram- negative short rods. However, the large amount of capsule surrounding Klebsiella these cells often interferes with the planticola destaining process, causing them to stain Gram positive. Endospores may be visible in older Bacillus subtilis cultures as clear areas inside the cells. Older cultures may stain pink, due to the deterioration of cell walls. This microbe forms gram-positive rods. Endospores may be visible in older Bacillus cereus cultures as clear areas inside the cells.
However, the large amount of capsule surrounding these cells often interferes with the Bacillus polymyxa destaining process, causing them to stain Gram positive. Staphylococcus This microbe appears as gram-positive epidermidis cocci. The Gram reactions of the 9 isolates. Colonies are punctiform, convex with Micrococcus luteus an entire margin and appear yellow on most medium Colonies are punctiform, convex with an entire margin.
The smallness of the Enterococcus colonies is due to the inefficient faecalis metabolism of these microbes. They can take days to reach appreciable size. Colonies are punctiform, convex with an entire margin. The smallness of the Lactobacillus colonies is due to the inefficient plantarum metabolism of these microbes.
Colonies are larger, but not as large as Bacillus subtilis B. The margin is undulate, with circular form and flat elevation. Colonies are large, irregular and flat Bacillus cereus with an undulate margin Colonies are large, irregular and flat Bacillus polymyxa with an undulate margin Staphyloccocus Colonies are circular, convex with an epidermidis entire margin Note the margin, shape and elevation of the test strains.
Also record the color of each strain. Figure Catalase reactions for test strains Culture Reaction Description Note the bubble formation. Catalase pneumoniae positive Note the bubble formation.
Catalase Serratia marcescens positive Pseudomonas Note the bubble formation. Catalase fluorescens positive Chromobacterium Note the bubble formation.
Catalase violaceum positive Note the bubble formation. This Enterococcus microbe uses peroxidase to detoxify faecalis H2O2, an enzyme that does not evolve oxygen. Note the lack of bubbles. This Lactobacillus microbe uses peroxidase to detoxify plantarum H2O2, an enzyme that does not evolve oxygen. Note the bubble formation. Catalase Bacillus subtilis positive Note the bubble formation. Catalase Bacillus cereus positive Note the bubble formation. Catalase epidermidis positive Note the catalase reactions of each of the test strains.
Look for evolution of bubbles. Klebsiella This microbe is negative for indole production. It will often give a false positive, as shown here, Serratia marcescens due to the extraction of cell pigment into the upper organic layer. Pseudomonas This microbe is negative for indole production.
Enterococcus This microbe is negative for indole production. Bacillus cereus This microbe is negative for indole production. Bacillus polymyxa This microbe is negative for indole production. Staphylococcus This microbe is negative for indole production. Note that only E.
Observe the bubble in the Durham tube. This microbe not only ferments glucose to Klebsiella acid, it also produces gas. Observe the bubble pneumoniae in the Durham tube. This microbe not only ferments glucose to Serratia marcescens acid, it also produces gas. Lactobacillus This microbe can ferment glucose to acid, but plantarum does not produce gas. Bacillus subtilis This microbe cannot ferment glucose.
Bacillus cereus This microbes can ferement glucose Bacillus polymyxa This microbe can ferment glucose Note the interesting pattern for this microbe. Staphylococcus Purple on the inside of the tube, yellow on the epidermidis outside. This is a positive reaction. Note the presence of growth, color and presence or absence of a gas bubble for each strain. Klebsiella Note the formation of acid yellow color and pneumoniae gas. Serratia marcescens This particular strain does not ferment lactose.
Pseudomonas The tube remains purple with no gas since the fluorescens microbe is unable to ferment lactose Chromobacterium This microbe is unable to ferment lactose. Enterococcus This microbe ferments lactose faecalis Lactobacillus L. Bacillus subtilis This microbe is unable to ferment lactose. Bacillus cereus This microbe is unable to ferment lactose.
Bacillus polymyxa ferments lactose and Bacillus polymyxa produces gas. Note the interesting pattern for this microbe. This microbe is Escherichia coli unable to hydrolyze starch and does not produce amylase. Note the lack of a zone of clearing. This microbe is Klebsiella unable to hydrolyze starch pneumoniae and does not produce amylase. This microbe is Serratia unable to hydrolyze starch marcescens and does not produce amylase. This microbe is Pseudomonas unable to hydrolyze starch fluorescens and does not produce amylase.
This microbe is Chromobacterium unable to hydrolyze starch violaceum and does not produce amylase. This microbe is Micrococcus luteus unable to hydrolyze starch and does not produce amylase. This microbe is Enterococcus unable to hydrolyze starch faecalis and does not produce amylase.
Lactobacillus plantarum does not grow well on starch medium and is difficult to see Lactobacillus in the photograph. Note the plantarum lack of a zone of clearing.
This microbe is unable to hydrolyze starch and does not produce amylase. Note the zone of clearing. All the starch in the medium Bacillus subtilis near the microbe has been hydrolyzed by extracellular amylases.
All the starch in the medium Bacillus cereus near the microbe has been hydrolyzed by extracellular amylases. All the starch in the medium Bacillus polymyxa near the microbe has been hydrolyzed by extracellular amylases.
This microbe is Staphylococcus unable to hydrolyze starch epidermidis and does not produce amylase. Observe for zones of clearing around starch plates that have been flooded with iodine. Note that the zone of clearing should be larger than 3 mm.
This microbe can Klebsiella reduce nitrate to pneumoniae nitrite. This microbe can Serratia reduce nitrate to marcescens nitrite. Pseudomonas fluorescens reduces nitrate all the way to nitrogen gas N2. This will cause the nitrate broth to not turn red when reagents are Pseudomonas added. Subsequent fluorescens addition of zinc, which will chemically reduce nitrate to nitrite, will not occur, since there is no nitrate to act upon.
Thus the tube will not turn red. This microbe can Chromobacterium reduce nitrate to violaceum nitrite. This microbe is negative for the nitrate test. Micrococcus Addition of zinc to luteus the test tube after adding reagents results in a weak red color. This microbe can Enterococcus reduce nitrate to faecalis nitrite This microbe is negative for the nitrate test.
Lactobacillus Addition of zinc to plantarum the test tube after adding reagents results in a weak red color. This microbe can Bacillus subtilis reduce nitrate to nitrite This microbe can Bacillus cereus reduce nitrate to nitrite This microbe can Bacillus polymyxa reduce nitrate to nitrite.
This microbe can Staphylococcus reduce nitrate to epidermidis nitrite. Reaction in nitrate broth for the various test species. Figure Motility of selected strains Culture Reaction Description This microbe is highly motile and will Escherichia coli show turbidity throughout the tube. Klebsiella This microbe is nonmotile. Note how the Serratia marcescens microbe swims to the top of the tube where there is more oxygen. This microbe is motile. Note how the Pseudomonas microbe swims to the top of the tube fluorescens where there is more oxygen.
Note how the Chromobacterium microbe swims to the top of the tube violaceum where there is more oxygen. Micrococcus luteus This microbe is nonmotile. Enterococcus faecalis This microbe is nonmotile. This microbe is nonmotile. It is often Lactobacillus difficult to see in the tube, due to low plantarum growth.
Note how the Bacillus subtilis microbe swims to the top of the tube where there is more oxygen. Note how the Bacillus cereus microbe swims to the top of the tube where there is more oxygen. Note how the Bacillus polymyxa microbe swims to the top of the tube where there is more oxygen.
Staphylococcus This microbe is nonmotile. Each is one of the ten microbes that you observed results for in the previous sections. By clicking on the button below, reactions in the various media will be presented to you.
Interpret the reactions and then using a table, or dichotomous key [32] that you generated from know results, determine the identify of your unknown. Get Unknown Isolate [33] 7 - 6 Antibody tests One important limitation to metabolic tests is that they almost always require the growth of the microbe in some medium [34] and this takes at least hours before a clear result is obtained.
Faster results would speed diagnosis. A second limitation to metabolic tests is that a specific isolate under testing may have a mutation [35] such that it is incapable of performing a metabolic conversion where the normal isolate of that species is proficient.
For example, most strains of E. Finally, to clearly identify an isolate, it is often necessary to perform numerous tests. The preparation and use of all this media is time consuming and expensive. Because of the limitations of metabolic tests, considerable effort has been made to find accurate, simple, and rapid alternatives. This research is starting to bear fruit, with the most popular methods being antibody [36] tests and DNA [37] methods, especially Figure [38] shows the steps of the TECRA reaction.
Here we present the TECRA reaction, explaining the theory behind how it works and a typical protocol for its use. The protocol was adapted from the technical material provided by BioTrace International. This kit will be used for various experiments depending upon time and funding. The protocol is the same for both after the initial enrichment steps.
Here is what is happening in each step 1. High affinity "capture" antibodies specific for the test microbe have been adsorbed on the surface of the Removawells. If the test microbes antigens are present in the sample, they are captured by the antibodies. All other materials in the sample are washed away. The sandwich is completed by the addition of enzyme-labeled antibodies Conjugate specific for the test microbe.
The presence of the test microbe is indicated when the bound conjugate converts the Substrate to a green color.
Alternatively, test microbe, no green color. If the test microbes antigens [39] are present in the sample, they are captured by the antibodies. The presence of the test microbe is indicated when the bound conjugate converts the Substrate [40] to a green color. Following enrichment, at least 1 CFU of the species of interest in 25 grams of sample can be detected. Presumptive positives should be confirmed by standard methods. This is especially important in situations such as product recalls.
This method has been shown to be at least as sensitive as standard culture and plating techniques and has virtually no cross-reactivity with other types of organisms. Performing the Immunoassay: 1. Transfer 0. Press the wells firmly into place in the holder.
NOTE: Thorough washing in these next steps is a critical step and is essential for a clear interpretation of the results.
Dump out the liquid in the Removawell. Remove residual liquid by striking the holder firmly several times face down on a thick pile of absorbent paper towels. This is important for effective removal of sample residue. Completely fill each well with wash solution, taking care not to trap air bubbles in the bottom of the wells.
Wash and completely empty the wells a total of 3 times as outlined above. Ensure the Removawells are empty before proceeding. Empty the wells and wash them thoroughly a total of FOUR times not three using the sequence previously described in step 7 Add ml of Substrate to each well.
Color development tends to concentrate around the sides of the wells. Tap the holder gently to distribute the color evenly before reading the result. Incubate for 10 minutes, then read the results. Results can be read visually using the Color Card provided OR with a plate reader. If using the card, compare your sample to the card. If using a plate reader, perform steps We will use the nm filter.
After 10 minutes, add ml of Stop Solution to each well. Tap the sides of the holder gently to mix the contents, then read the result. For the test to be valid: The positive control must have an absorbance of at least 0.
A sample is considered negative when the assay has proved valid and the sample well has a reading of less that 0. A sample is considered positive when the assay has proven valid and the sample well has a reading greater that or equal to 0.
The results can be observed qualitatively using a card or quantitatively using a plate reader. All enzymes are encoded in the genome the DNA [42] of the microbe. It is therefore possible to detect the presence of the genes that codes for specific enzymes instead of detecting the metabolic products of that enzyme activity. DNA detection methods depend upon the hybridization [43] of a short piece or pieces of synthesized DNA to the genome of the test species. These short pieces of DNA are called probes or primers depending upon the method.
The DNA genome of the test species is extracted, often by simply boiling a small bit of culture of the microbe, and then exposed to the primer [44]. DNA detection has several advantages. Depending upon how the primer is designed and what the target is, the test can be specific to a species or even a subspecies, or it can detect multiple microbes.
For example, a primer can be made to the shigga-like toxin of certain strains of E, coli, specifically identifying heamorrhaggic E. On the other end of the spectrum, there are primers that have been created to conserved regions of the 16S rRNA [45] of bacteria [46] that will detect any bacteria present in a sample. A mutation [47] that would inactivate an enzyme, and fail in a biochemical test, will often still be positive in an DNA detection method.
With a well designed probe [48] or primer a single DNA test can identify a microbe. Identification by metabolic tests requires a large number of tests to narrow it down to the species level.
Until a few years ago, detecting the presence of specific DNA sequences was much more difficult than performing metabolic tests. Even though there are advantages to using DNA tests, they remained something done in research labs for very different purposes. Microbes can be detected at very low levels, sometimes just a few cells per gram [49] of sample are detectable. Real time PCR is a modification of the PCR protocol such that PCR fragments are rendered detectable by fluorescence, thus it become unnecessary to run an agarose gel [50] to determine the absence or presence of a DNA fragment.
Many kits are available to detect pathogens in food. However, UW-Madison uses a modified protocol that simulates the results one would obtain from a kit. Why are we not using a kit? Two reasons. Second, they have very high specificity. For example, the kit for hemorrhagic E. Therefore, only the bona fide pathogen [51] will work in the assay. While this is a good thing in the field, in a teaching laboratory it would require that you be given the real pathogen; probably not a good idea.
Especially with hemorrhagic E. In our assay, fluorescence is generated by the binding of SYBR green, a dye developed by Invitrogen [53]. The dye is highly specific for double stranded DNA, and has a much higher fluorescence when bound to it. If a positive template is present, a strong SYBR green fluorescence will be observed in the tube. Conversely, samples without a positive template will have a lower fluorescence. It is therefore important to perform a melting curve after the amplification to verify that the fragment obtained is the correct sequence.
DNA sequences will have a unique temperature at which they melt, with the melting temperature dependent upon the specific primary sequence of the fragment. In most RT-PCR reactions, the correct fragment will have a unique melting temperature compared to any false positives that may have arisen in the tube.
With a carefully designed experiment and given the right primers, it is possible to test for the presence or absence of any DNA sequence and therefore any desired microbe or set of microbes. This protocol was adapted from the references listed at the end of this page.
Procedure For a food or other natural sample, perform the following. The sample must first be washed and centrifuged at low speed to remove large particulates before harvesting the microbes.
Resuspend 0. Vortex vigorously for about 30 seconds. Spin the sample at x g for 15 minutes. Carefully pipette off the supernatant into an Oakridge centrifuge tube and place the tube on ice 3. Repeat steps 1 and 2 two more times on the original sample, pooling approximately 9 ml of supernatant. For a colony to be tested, perform the following 1. For a broth or enrichment culture 1.
Centrifuge at 14, rpm or at high enough velocity to pellet the cells for 20 seconds. Once processed continue from here 1. Chill the sample on ice for 10 minutes. Centrifuge in a table top centrifuge for 1 minute and collect the supernatant. This liquid will serve as your source of template DNA.
Prepare your real time PCR tube according to the following table. Master mix is purchased from Promega [57] corporation. It comes as a 2x concentration. The SYBR green dye is purchased from invitrogen. It is used in PCR assays at , x dilution.
A 10x solution is a 10,fold dilution of the SYBR green. The primers are used as a concentration of primer is 0. We obtained the primers from genosys [58]. References 1. J Food Prot. The system is programmed using their software. Runs typically take about hours to complete. Results consist of Amplification plots c and Melting Temperature curves d. Final analysis consists of looking at what cycle fluorescence appeared and what the melting curve of each test looks like in comparison to the positive and negative control.
Cycle threshold indicates the cycle at which the fluorescence detectedby the instrument passed a predetermined threshold, indicating the presence of a PCR product. Melting temperature is the temperature at which the amplified DNA double helix denatured into single strands. This is sequence specific. Any test PCR reaction should have approximately the same melting point as the positive control.
Given this table of results, determine which of the 14 samples actually contained E. For more information on identifying and classifying bacteria, read the chapter on bacterial classification [60].
Learning the members of a population of microbes present in nature can give us new insights into biochemical processes that are taking place. This leads to a better understanding of environments and the role microbes play in them. Identifying a microbe growing in a patient will identify the disease and the treatments that are effective at eliminating it. Identification tests can also be used to monitor and eliminate microbes present in the food supply, increasing food safety by eliminating pathogens and decreasing spoilage.
These are just a few of the many reasons for identifying microbes. For decades, microbial identification had dependent upon determining the biochemical capacity of the microbe by growing in various test media.
These tests probe [61] the metabolic capacity of the strain under study, determining what the microbe could use as a carbon source fermentation [62] broth , its relationship to oxygen thioglycollate medium [63] , cell wall structure Gram [64] stain , and many other properties. Hundreds of media and tests have been developed to help identify microbes. These tests can be fairly accurate, but because many depend upon growth of the microbe, they often require a one day incubation before they can be read.
This can be a serious detriment, especially in the food and health field, rapid diagnosis is especially critical. A search for more rapid methods lead to the development of tests based on antibodies and DNA [65] methods.
Antibody [66] methods depend upon the reaction of a specially prepared antibody against an antigen [67] that is unique to the target microbe. The most common DNA methods utilize the short DNA sequences called primers that hybridize to distinct sequences in the target microbe. The primers are then used as a template [68] in PCR reactions, producing a detectible PCR fragment, that indicated the presence of the target microbe.
Antibody and DNA-based methods are more rapid than classic biochemical tests, but they tend to be specialized for the presence of specific microbes. Due to the large body of information that has been gathered on most cultured micorbes, it is now possible to design a set of tests to determine the identity of almost any microbe. Chapter 8 - An Introduction to Bacterial Genetics 8 - 1 Definitions in bacterial genetics One of the major goals of genetic studies is to understand the organization and function of genes.
To achieve this, alterations in DNA [1] are created, either spontaneously or by the experimenter, fished out in some fashion, and the effects of these changes are observed. It is analogous to systematically breaking the pieces of a radio, seeing what happens, and from this information deducing the function of each component.
Bacterial genetics has helped to unravel the mysteries of DNA replication, the production of proteins from DNA, the genetic code, gene regulation and more! Genetic analysis will impact your life in many ways. The human genome project which will sequence all 23 of the human chromosomes involves the use of sophisticated bacterial genetic techniques. A growing list of mammalian and other proteins that are naturally found in extremely low amounts, are being expressed in microorganisms and purified including 1 human insulin-produced in bacteria [2] for diabetics, 2 human growth hormone [3] for treating dwarfism, and 3 proteins for the production of vaccines [4] against diseases.
On a more familiar level, bacterial genetics is being employed to improve the strains used to make cheese, beer, yogurt, and other fermented food products. Here are a few key terms to help you understand bacterial genetics. A mutant [5] is a strain that has an altered growth property termed the phenotype [6] when compared to a designated bench mark strain, which is referred to as the wild-type strain [7] wt.
A mutation [8] is an alteration in the DNA sequence of an organism. Some mutations will cause a detectable change in its phenotype, others will not and are called silent mutations. The genotype [9] of a microorganism is its DNA sequence. For example, if the wild-type [10] strain is mutated so that it is unable to synthesize its own tryptophan, it is referred to as a tryptophan minus mutant its phenotype is termed Trp-.
Its genotype is the specific base [11] pair change that has taken place and this can be determined by DNA sequencing. If this mutant is plated onto minimal medium [12] lacking tryptophan, most of the organisms will be unable to grow. These are referred to as revertants [14]. They can arise from a conversion of the mutant genotype back to wild-type, or from another secondary mutation that counteracts the initial mutation. Do not confuse a revertant with the wild- type strain, since reversion [15] can also be caused by secondary mutations.
For example a Trp- mutant is termed a tryptophan auxotroph. A prototroph [17]is an organism not requiring the nutrient in question, a wild-type strain is prototrophic.
For more information, a good microbiology textbook will have a chapter on Bacterial Genetics [18]. Touch a sterile loop to the edge of the area and streak the plate of MacConkey Agar for isolated colonies. Potato knives 1 sterile petri dish with sheets of filter paper 1 tube of sterile water 5 ml 1 culture of Escherichia coli Figure E. For your group of people, obtain a potato from the ethanol bath; handle only at the ends. Place on a fresh paper towel. Place your slice into the sterile petri dish.
Moisten the paper in the dish with the sterile water. Examine your Mac plate, E. The red color is caused by the fermentation of lactose, a resulting drop in pH, and an accumulation of the red dye, neutral red. Make careful note of the colony appearance. Would this appearance automatically cause the organism to be termed a coliform? Inoculate one cut with a suspected E. Inoculate the other cut with a loopful of the E. Observe the potato slice for evidence of soft rot.
Describe the symptoms. Are they similar to what you saw in the original infection? Touch a sterile loop to the edge of the soft rot and streak the MacConkey Agar plate for isolated colonies. Period 4 Figure E. If present, this supports Koch's postulates and indicates E. Picture of second MAC plate. Observe the colonies on the MacConkey Agar plate. Do the colonies appear to be the same type observed previously?
How do the foregoing procedures and observations fit in with Koch's Postulates? Assign each task you have done in this protocol to a postulate. Links 1. You have studied some of the physiology of bacteria [1] as they relate to culture media and biochemical testing. You probably know where in this manual you can find information on specific bacterial characteristics and the procedures for the various tests.
The purpose of this exercise is to correlate the various bits of information to strengthen your understanding of, and familiarity with 1 the media and tests used this semester and 2 the major characteristics of the genera of bacteria with which you have had some experience.
We have been using typical species of these genera throughout the course. Dichotomous Key The exercise will consist of two parts. First, you will prepare a Figure [2] and Figure 2 [3].
Use tests such as the primary tests considering any of the secondary tests! Some things to consider in making your key: 1. If you believe that either a primary test e. The only instance where lactose fermentation was for us of realistic importance in differentiating between genera was in Experiment 14 for the enteric bacteria [5]. Can glucose fermentation be used to differentiate Bacillus from anything else such as Lactobacillus?
Go back to Section [6] and note that we studied several species of Bacillus - some of which were facultative anaerobes [7].
That should answer the question. Where you may have had some experience with an organism having a variable reaction or a weakly or delayed positive reaction e. Use something more definitive! Where you may not know a reaction for a particular organism H2S for Bacillus? Glucose fermentation for Neisseria? One does not have to know all of the reactions for the 12 organisms to make a dichotomous key.
With these things in mind, you can prepare a key which will be of use in characterizing typical members of these genera, even outside of the course. This key should be submitted no later than the two weeks before the course ends. It will then be graded and handed back in time for you to consider which tests you will need to identify your unknowns. Isolation and Identification of Unknowns The second part of this endeavor involves the actual isolation and identification of the three components of the mixed unknown.
One typical representative of each of three genera on the list below will be found in this mixture. Naturally you should want to start off by checking the mixture with a gram [8] stain and then streaking for isolated colonies [9]. Note that you have two media for this purpose. One - Heart Infusion Agar - is an all-purpose medium [10] which will support the growth of all 12 genera.
The other - MacConkey Agar - is a selective- differential medium which will in this experiment support the growth of only the gram- negative rods and will differentiate between any two or more different organisms growing on it. Which may be best to prevent "overgrowth? Remember to record your unknown number. Figure Possible organisms in the test Bacillus Staphylococcus Neisseria Micrococcus Morganella Enterobacter Pseudomonas Klebsiella Citrobacter Lactobacillus Escherichia Enterococcus The table shows the possible genera of the species of microbes that you may find in this test.
You must design a dichotomous key to help you work out the identity of your microbe. You may use any of the media or reagents listed here to identify your isolate. This is more than sufficient and please don't ask for media or reagents that are not on this list.
Don't forget the Gram stain! How old should the culture be? Can a reliable Gram stain be made directly from a MacConkey Agar colony? What about oval cells? Are they rods or cocci? Remember that the Gram stain and the catalase and oxidase tests should be run only on cultures growing on all-purpose media.
A word of caution: Do not identify anything as Neisseria unless 1 it is oxidase-positive and 2 it looks exactly like the Neisseria slide from Experiment 12 [11] The teaching personnel will be available if you hit a snag.
Do not expect them to lead you through the procedure! You are on your own! Answers to most or all of your questions will be found in your notes and experience or somewhere in this manual. They were enthusiastic, friendly, patient with the annoying instructor who was always stealing their plates for photos, technically adept, and more than willing to help John, Loretta and myself create the hundreds of photos and movies that make up this book.
As a final act of gratitude, I list all their names. Streptomyces and molds are especially equipped with extracellular enzymes to hydrolyze the starch and casein to obtain carbon, nitrogen and energy. Potassium nitrate serves as an supplemental nitrogen source. Sodium propionate is added to discourage growth of molds. The inoculated medium is overlaid with several ml of sterile mineral oil such that the organisms will respire all of the oxygen in the medium now sealed against oxygen entry and render it anaerobic [7].
Fermentation [8] of the glucose to acidic products will result in the bromcresol purple turning yellow unless decarboxylation of the amino acid also occurs, in which case a net alkaline reaction purple color will be seen.
For each organism tested, a control tube of the basal medium i. Failure to achieve an acid reaction in the basal medium means that the organism is probably a strict aerobe therefore not an enteric and that results in the test media are meaningless. Lactose is a carbon source used by all coliforms and bile salts selects against gram-positive bacteria. Used in the confirmatory test for total coliforms; see Experiment 15 [10].
The medium is especially selective because of the high temperature.. With rare exceptions, fecal coliforms are the only organisms which will not only grow in this medium but also produce gas from lactose fermentation at Used in the confirmatory test for fecal coliforms; see Experiment 15 [11]. Used in the completed test for isolation of individual coliforms to be identified by IMViC and other tests. Eosin Y and methylene blue serve as selective agents, inhibiting gram-positive bacteria.
These materials also serve as the pH indicator [12]. Lactose-fermenting colonies will produce acid, resulting in a dark color. Non-lactose-fermenters produce pinkish or lavender colonies. See Experiment 15 [13]. Occasionally as an all- purpose broth medium. Also used as a base [14] for Thioglycollate Agar and Thioglycollate Medium. Cystine and sodium thioglycollate are included in the medium to maintain a low oxidation-reduction [16] potential.
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