Welcome to the Clinical Laboratory MICROBIOLOGY Microbiology Lab Is this evidence of an infection? Which colony is the “important” one? Why does the bacteria causing disease need to be identified to treat the infection? Why not just give antibiotics? These are questions asked and answered by the microbiology lab. Do you want a footer? Investigation of disease in the Clinical Microbiology Lab In a clinical microbiology lab, experienced clinical microbiologists begin their investigation of disease by using microscopy to examine smears of original samples to obtain important early information. The arrows are pointing to bacteria…but the identification is near impossible at this point. Do you want a footer? General Sequence of Events in Identifying Bacteria List of steps toward identifying the cause of infection: 1) Microscopic examination of specimen to determine if there’s bacteria present 2) Gram stain to determine type of bacteria 3) Growth of bacteria in broth 4) Plate broth bacteria on media plate (culture) 5) Select isolated pathogen 6) Perform biochemical testing 7) Perform antibiotic susceptibility Thoughts on Identification of Bacteria Maybe we need to go back a bit. The entire reason to identify a bacteria that we suspect is causing disease is to be able to treat it. Bacteria is almost always treated with antibiotics – but they must be specific to the bacteria. One antibiotic won’t work for all bacteria. When a specimen arrives in the micro lab, it’s looked at microscopically and gram stained, but then it needs to grow outside the body so that we can work with it. o you want a footer? Thoughts along with Videos… Video on Gram staining (the lady in this video refers to their lecture and lab manual – don’t be confused, these are theirs, not yours!) Gram Stain Video Inoculating a broth with the bacteria is almost always the first step in growing it. Video on isolating bacteria Isolating Bacteria Thoughts… Once there’s sufficient growth (8-12 hours) the bacteria present is put on a petri dish of media. There is likely a number of bacteria in the broth, the pathogenic ones need to be isolated out. This process sometimes requires that steps be repeated because the bacteria to be tested needs to be absolutely pure. Do you wnt a footer? Gram Stain After looking at the specimen under the microscope, a Gram stain is done. The Gram stain is a technique for staining and detecting bacteria and yeasts. It is the most commonly performed procedure in the clinical microbiology laboratory. Four reagents are used to perform a Gram stain: crystal violet, Gram's iodine, acetone-alcohol, and safranin. Gram Stain Grams stains are used to differentiate types of bacteria. Gram-positive and Gram-negative bacteria stain differently because of the structure of their cell walls. Gram negative = red or pink Gram positive = purple Gram Stain Gram stain results can have a dramatic effect on patient care. Hospitalization may be required when the Gram stain indicates bacteria are present in a normally sterile body fluid. Sterile body fluids include blood and CSF. It’s very dangerous to have bacteria in these fluids. Remember, the point of all this work is to identify the pathogenic bacteria so that it can be treated. The initial choice of antibiotic therapy can be guided by the Gram stain results. Do you want a footer? Gram Stain Once a gram stain identification is made a culture plate (or petri dish) is set up so that the bacteria can grow. Gram Stain Video Gram Stained Bacteria Another use of Gram stain is observing the arrangement of its colonies as it grows. Both of these characteristics are helpful when identifying bacteria. Gram Positive Cocci The Gram-positive coccus (singular) is a spherical bacterium. Gram-positive cocci may appear in four clinically significant groupings: dipplo (two cells growing very close to one another), chain, tetra (4 cells close), cluster. Gram positive dipplococci for example is frequently Streptococcus pneumoniae. See how these cells are growing in pairs? That’s what dipplococci look like. Another example of the importance of knowing the shape and arrangement of bacteria would be the differences seen between Strep. anginosus which grows in chains and Staph. epidermidis. While both are gram positive cocci, S. anginosus is often the cause of oral infections and very pathogenic. Staph. epidermidis is found in great numbers on our skin and is not pathogenic at all. See how the arrangement of cells look different? Gram Positive Bacilli The Gram-positive bacilli is a rod shaped bacteria. Gram-positive bacilli may also appear in four clinically significant groupings... Long, wide Long, narrow, often chaining Coccobacillius Branched Gram Negative Cocci Gram-negative cocci may appear in two clinically significant groupings... Cocci Dipplococci Gram Negative Bacilli The Gram-negative rod or bacillus is a rectangular shaped bacterium. Rods are variable in length, width, and staining characteristics. There are five clinically significant shapes of Gram-negative rods... Technologists in Micro use these adjectives to describe what they see on the Gram stain. because these descriptions are used universally, the doctor has a good idea about the identification of the bacteria. Long, narrow Coccobacilli Curved rod Fusiform Spiral rods Colony Morphology Once the bacteria is grown in a broth, it is put on a specific media in a petri dish, or culture plate. Cultures are generally incubated at 37◦ C in special atmospheres to maximize growth of pathogens. Along with the arrangement of growing colonies, colony morphology, or the shape of a colony after growth on the plate is often diagnostic. Colony Morphology Common morphologies: Colony morphology This is an interesting picture because it shows the same bacteria Gram stained and on the plate. Describing Bacteria If we were going to describe the isolated bacteria on the preceding slide we might say something like, “Gram positive bacilli, small, round, slightly mucoid (wet looking), slightly raised colonies.” Colony morphology So, the technologist actually uses these observations to describe and identify disease causing bacteria. Some bacterias hemolyse blood in this media (BAP media) during growth. The degree of hemolysis can be used to describe and identify the bacteria. This is a great illustration of three types of hemolysis: alpha hemolysis, beta hemolysis, and gamma hemolysis. Using these two images and the following descriptors, describe the bacteria seen here Gram stain Morphology Colony type Hemolysis - Mixed culture Pure culture Pathogenic bacteria Often the source of the specimen determines if the bacteria is pathogenic. For example, Streptococcus pneumoniae is a pathogen in the blood, but considered a normal inhabitant of the throat. Normal inhabitants are called normal flora. The gut has thousands of species of bacteria as normal flora. Techs need to know where specific bacterias should be (therefore normal) or shouldn’t be, (therefore pathogenic). Biochemical Identification One of the most distinguishing features of bacteria is their biochemical versatility. The types of biochemical reactions each organism undergoes acts as a "thumbprint" for its identification. This is based on the following chain of logic: Do you want a footer? Biochemical Identification Each different species of bacterium has a different molecule of DNA (i.e., DNA with a unique series of nucleotide bases). Since DNA codes for protein synthesis, then different species of bacteria must, by way of their unique DNA, be able to synthesize different protein enzymes. Enzymes catalyze all the various chemical reactions of which the organism is capable. This in turn means that different species of bacteria must carry out different and unique sets of biochemical reactions. Biochemical Identification The battery of biochemical tests used is dependent upon the gram stain of the bacteria. Testing can be manual or automated. This panel is read on an instrument using something very similar to spectrophotometry. Antibiotic Susceptibility Once the pathogenic bacteria is identified, the doctor will want to know how to treat the patient. Obviously, certain antibiotics are more effective against some bacterias than others. They are often chosen by the degree of their selective toxicity. The selective toxicity of antibiotics means that they must be highly effective against the microbe but have minimal or no toxicity to humans. Antibiotic Susceptibility Remember that certain bacteria are normal and even helpful in certain locations. If normal flora is killed, health is compromised. This makes selecting the right antibiotic at the right does very important. Antibiotics That Work Antibiotics are chosen depending upon the gram stain of the bacteria. In other words, an antibiotic will be effective against a gram positive bacteria, but not against gram negative bacteria. Discs with particular antibiotics are placed on a plate with a pure culture of the bacteria. Antibiotic Susceptibility Discs with antibiotic The antibiotic works if there is a zone of inhibited growth. These zones are measured giving the doctor a clue to which antibiotic may be most effective. Would this antibiotic be effective against this bacteria? Antibiotic Susceptibility Manual testing Automated susceptibility testing Microbiology lab All of this testing in the micro lab generally takes only 48 hours. Some rare labs, in large hospitals, are using PCR for screening of disease-causing bacteria that is highly contagious. This enables the lab to report the identification of the bacteria in less than 12 hours. Review Questions True or false: The first thing done when a specimen is received in micro is the Gram stain. What color is a Gram negative bacteria? What color is a Gram positive bacteria? Why does pathogenic bacteria need to be isolated? Define normal flora. Briefly explain the benefit of doing biochemical tests on unknown bacteria. What is the purpose of performing antibiotic susceptabilities?