LAB GEN MICROBIOLOGY
LAB GEN MICROBIOLOGY BIOL 208
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IAEORATORY WEEK 10 EXAM 11 Ex 14 25 208 MICROBIOLOGY SECTION 3 rmsxowcxou mmmmus or ox mm rmnzxonkmmocms 7 man u 1051mm mmmm or 4 mvoxmrxouymrmm 4x mus UnuxmcmmomqumA I momm r m u wmsm W n iembvc WWW y chincmnxed by the meme m m oxykn 02 We m madam 1mm Vick m oxygen mm mm mm oxygen bound m WWW m mmm men mmmmo mammxsqx m m mvcmbe m humanva mm m bu n mqumq N u m ibmmd form mm vmmid There vxby de mnun byvmaumxmmbm common the mumskn mm mm 0 omzwhere when the oxygen imm y bvvchem y m mquot nmvedby memvcmbe m Iv e mu mum mmm m Wm m e civil91 Wm iembe and m m ibxevme of oxykn y weerth urge mm why u lembvc rexvvnnun mm m WWW may mm q vmvvde much mm mmmmm vmm and y enugenca y wnd Gram mm enduivun mm rad EXERCISE 3 1 ENDOSPORULATION PATTERNS Clostridia are obligately anaerobic spore forming straight to slightly curved bacilli that usually stain Gram positive Spores of the organisms are ubiquitous in soil are found in the intestines of animals including man and are present in marine and aquatic sediments Clostridium difficile is now recognized as the major causative agent of coIitis in ammation of the colon and diarrhea that may occur following antibiotic intake C dif ciIe infection represents one of the most common hospital nosocomial infections around the world In the United States alone it causes approximately three million cases of diarrhea and colitis per year This bacterium is primarily acquired in hospitals and chronic care facilities following antibiotic therapy covering a wide variety of bacteria broad spectrum and is the most frequent cause of outbreaks of diarrhea in hospitalized patients One of the main characteristics of C dif cile associated colitis is severe inflammation in the colonic tissue mucosa associated with destruction of cells of the colon colonocytes The disease involves alterations in the population of the beneficial bacteria which are normally found in the colon by antibiotic therapy The alterations lead to colonization by C dif cile when this bacterium or its spores are present in the environment In hospitals or nursing home facilities where C dif cile is prevalent and patients frequently receive antibiotics C difficile infection is very common Clinical Features A wide range of conditions is associated with C dif cile infection Most cases develop 4 to 9 days after the beginning of antibiotic intake Although nearly all antibiotics have been implicated with the disease the most common antibiotics associated with C difficile infection are ampiciIIin amoxiciIIin cephansporins and cIindamycin Pseudomembranous colitis represents the characteristic manifestation of full blown C dif cile associated colitis Sigmoidoscopic examination reveals the presence of characteristic plaque like pseudomembranes scattered over the colonic tissue The presence of these plaques is a distinctive indicator of C difficile infection in patients with diarrhea following antibiotic treatment Laboratory studies show that when C difficile colonize the gut they release two potent toxins toxin A and toxin B which bind to certain receptors in the lining of the colon and ultimately cause diarrhea and in ammation of the large intestine or colon colitis Diagnostic protocols Current protocols target the presence C difficile toxin The Toxin A B test serologically detects both toxins A and B within 20 minutes A Diluted stool sample is mixed with conjugate and added to a test cassette for rapid identification Laboratory Week 13 Clostridium botulinum Botulism is a neuroparalytic disease produced by the neurotoxins of C botuIinum Seven distinct toxin types are produced by the organism A B C D E F and G Types A B and E are most commonly associated with disease in the United States 11 2 FACT Neurological symptoms and signs dominate the clinical syndrome of botulism Incubation periods for foodborne botulism are reported to be as short as 6 hours or as long as 10 days but generally the time between toxin ingestion and onset of symptoms ranges from 18 36 hours Clinical Features Dryness of the mouth inability to focus to a near point and diplopia double vision are usually the first complaints If the disease is mild no other symptoms may develop and the initial symptoms will gradually resolve In more severe cases the initial symptoms may be followed by voice impairment dysphonia difficulty swallowing dysphagia and peripheral muscle weakness If illness is severe respiratory muscles become involved leading to respiratory failure and death unless supportive care is provided Death occurs in 5 10 of cases of foodborne botulism THREE TYPES OF BOTULISM 1 Foodborne botulism has long been recognized in the United States It is often associated with improperly home canned vegetable products typically green beans red beets peppers mushrooms or asparagus Large outbreaks of botulism have been associated with restaurants using improperly canned foods or mishandled food products Strict adherence to appropriate processing methods assures the destruction of all bacterial spores and prevention of germination and outgrowth of spores in the product 2 Wound botulism is the rarest form of the disease Most cases involve infections in traumatic wounds contaminated with soil although cases have occurred due to a necrotic bowel or associated with chronic drug abuse The toxin is elaborated by the organism growing in a wound site 3 Infant botulism is the most common form of the disease in the United States The disease results from absorption of toxin produced by the toxigenic organisms colonizing the intestinal tract of certain infants under 1 year of age disease in older toddlers is rare The lack of established normal gut ora that would inhibit the germination of ingested spores and outgrowth of organism Clinical Features for Infants The child may be listless which resembles meningitis Toxins produced in the gut will result in constipation not feeding and oppiness oppy babyquot Clostt39idium tetani Tetanus is a condition also referred to as lockjaw CIostTidium tetam39 are Gram positive spore forming rods that are anaerobic If they enter the body through a wound they can multiply and produce a toxin that effects the nerves and controls the activity of muscles C tetam39 spores can be acquired from any type of injury involving an infected device Being punctured by a rusty nail is a common the source of an infection but infections can also occur from a wound a burn an ulcer a compound fracture operative wounds aquired during operations or a drug injection If an anaerobic environment is present the spores will germinate and form active cells If not treated early the death rate of C tetam39 is high After being infected the first sign is a lesion in the skin that is usually not noticed The spores germinate and the new cells release a toxin called tetanospasmin Usually it will lie dormant for a few days to weeks but the shorter the incubation period usually the higher the mortality The muscles will begin to intermittently contract around the site of entry The toxin then passes retrograde along nerve bers and xes to nerve tissue 11 3 The toxin39s main target is the area around the brain stem Lockjaw occurs and is followed by general rigidity of the body then muscle spasms occur throughout the body Finally death is caused by interference with muscles required for breathing Luckily it is usually prevented through immunization FACT Neonatal tetanus continues to be an important cause of neonatal morbidity and mortality in developing countries A number of factors contribute to the high incidence of tetanus and infection in these countries most deliveries take place at home often in unhygienic circumstances deliveries are conducted by untrained birth attendants and some traditional cord care practices are harmful C perfringens is most frequently responsible for released toxins and enzymes that cause tissue damage These conditions are known as anaerobic cellulitis myonecrosis or gas gangrene Other clostridia that are occasionally responsible include C novyi and C septicum Gas gangrene nearly always starts in a wound usually in a limb with an anaerobic or dead tissue area This may be muscle or other tissue damaged at the time of the injury or a large blood clot Next this susceptible area must be contaminated with clostridial spores Then if the oxygen tension is low enough the spores will germinate revert to their vegetative state and begin to multiply When clostridia are established in a wound their enzymes and toxins begin to spread outwards killing more tissue This enlarges the anaerobic area into which the clostridia can spread Gas is produced by bacterial enzymes as they attack the tissues adding to the pathology as it splits muscles apart to allow the infection to extend When the pressure of the gas rises above the arterial pressure the blood supply to the muscle is cut off and the tissue dies This happens more easily because the blood pressure is likely to be low as a result of the original injury and it is reduced still further as the absorption of toxins leads to septic shock When these factors combine gangrene can spread to a whole limb in a matter of hours and death soon follows Another disturbing factor is when touched the offending area will sound like the crinkle of cellophane In addition to gas gangrene C Qerfringens is a common cause of food intoxication usually associated with 39 1 meat dishes Spores that survive the normal cooking process germinate as meat cools and within a few hours sitting at room temperature massive numbers of the bacteria have developed After ingestion the bacteria continue their growth in the intestines where toxins are released These toxins cause diarrhea cramps and abdominal pain Onset is eight to twenty hours after ingestion Symptoms only last for a day or so and death rarely occurs Control Thoroughly cook foods which contain meat and poultry soups stews gravy dressing casseroles Keep these cooked foods hot at or above 60C 140 F or cold at or below 4C 40 F ENDOSPORE LOCATION The location ofthe endospore is a genetic characteristic that helps with the identification of the organism In this exercise you will observe prepared slides of C batuinum sub terminal endospores and prepare a Gram stain smear of C sporogenes terminal endospores see figure 111 11 4 IHGURE11JENDOSPORES LEFT TO RIGHT TERMINAL CENTRAL SUBTERMINAL DC Procedure Step 1 Observer the prepared slide of C botuIinum Record staining reaction and endospore location Step 2 Observer the prepared slide of C tetam39 Record staining reaction and endospore location Note Clostridial cells are Gram positive when stained between 12 24 hours old These cells rapidly become Gram variable with age however in order to observe endospore location you have been given a 4 day old culture Expect some level ofgram staining variability ie cells will stain red gram negative instead of blue gram positive EXERCISE 3 2 W The following specimens Abscesses Pus Wounds Skin Tissue and Fluids are collected and transported so as to minimize contamination by normal aerobic and anaerobic ora The standard regiment for these specimens in most clinical laboratories would inclulde 1 Blood agar 510 COZ 37C 2 Blood agar anaerobic 37C 3 MacConkey agar aerobic 37C 4 Chocolate agar aerobic 37C 5 Thioglycolate broth 37C 11 5 Specimens from the following sites are acceptable when submitted in the appropriate anaerobic transport media Transtracheal aspirations Suprapubic urines Genital specimens from cul de sac aspiration placenta fallopian tube septic abortion or prostatic or seminal uid Surgical specimens Exudates aspirated pus from deep wound or abscesses Body uids normally sterile The following sites should not be cultured anaerobically because normal anaerobic ora is present Throat and nasopharyngeal swabs Sputum and bronchoscopic specimens Feces and rectal swabs Voided or catheterized urines Specimens from sites contaminated with intestinal contents such as colostomy sites All anaerobic specimens should be collected to minimize contamination with normal ora Specimen collection by needle aspiration is recommended when possible advertisement for Sioux Valley Clinical Laboratories ANAEROBIC SYSTEMS FOR CULTIVATION OF ANAEROBIC BACTERIA ANAEROBIC IAR TECHNIQUES The basic principle of all anaerobic jars is the same Removal of oxygen from the chamber by reaction with hydrogen added to the system in the presence of a catalyst The oxygen combines with H2 gas and is reduced to water The disposable GasPak hydrogencarbon dioxide generator system by BBL is a convenient way to carry out the reduction of oxygen and simultaneously replace it with a C02 atmosphere The Gas Pak package contains sodium borohydride and sodium bicarbonate that react upon addition of 10 mL of water to produce hydrogen and carbon dioxide gases The Gas Pak also contains a small packet of palladium coated alumina pellets to act as a catalyst in the reduction of oxygen 11 6 FIGURE 112 ANAEROBIC JARS W The BIOVBAG Type A Is a transparent rndmdual cusposable envtronrnental chamber that contarns a gas generator eonsrstrng of one tablet of potassrurn bomhydnde rsodlum ht rh n t When the BIOVBAG contarnrng all three components has been properly heatsealed and the generator aetwated an anaerobre envtronrnent W111 be created An oxygen reduetron rncheator resazunn rnonrtors the oxygen level Frgure113 FIGURE 113 BIOBAG DISPOSABLE ENVIRONMENTALCHAMBER 1177 TH IOGLYCOLATE BROTH The use of thioglycolate broth permits growth of anaerobic bacteria In addition growth patterns can help distinguish aerotolerance of bacteria Thioglycolate broth is a nutritive medium with a reducing agent sodium thioglycolate which removes oxygen from the broth A chemical indicator is included in the broth in this case methylene blue The blue color sometimes greenish color indicates the presence of oxygen Color plate 111 CULTURES Clostridium perfringens Thioglycolate Broth Staphylococcus epidermidis Nutrient Broth Pseudomonas aeruginosa Nutrient Broth W We will conduct steps 1 2 and 5 from the standard regiment listed at the bottom of page 115 Step 1 Label 2 Blood Agar plates one plate will be titled Aerobic and the other titled Anaerobic Aerobic Anaerobic Step 2 Using your inoculating loop streak each plate with Clostridium perfringens Step 3 Place the anaerobic Blood Agar plate in a Bio Bag or Gas Pac Jar The instructor will demonstrate proper set up of the enVironmental chamber Step 4 Incubate both plates at 37C for 24 48 hours Step 5 Obtain Three Thioglycolate Broth tubes 7DO NOT SHAKE THESE TUBES Gently label each Thioglycolate Broth with one of the assigned species being careful not to tip the tube horizontally You may use the order 12 and three as in Results table 1 11 Step 6 Using your inoculating loop inoculate each Thioglycolate Broth with its assigned species Step 7 Incubate the Thioglycolate Broth at 37C for 24 48 hours 11 8 RESllLII ABLILLLl GRAM STAINING AND ENDOSPORE LOCATION ASSIGNED SPECIES GRAM REACTION DIAGRAM MORPHOLOGY AND ENDOSPORES C b0 tuIm um C tetam CULTURING ANAEROBIC BACTERIA AEROBIC BLOOD AGAR PLATE ANAEROBIC BLOOD AGAR PLATE C perferin gens S epidermidis P aeruginosa 11 9 MYCOBACTERIA Mycobacterium tuberculosis is the etiologic agent of tuberculosis TB in humans Humans are the only reservoir for the bacterium Mycobacterium bOVis is the etiologic agent of TB in cows and rarely in humans Both cows and humans can serve as reservoirs Humans can also be infected by the consumption of unpasteurized milk This route of transmission can lead to the development of extrapulmonary TB exemplified in history by bone infections that led to hunched backs Tuberculosis commonly called TB is a contagious disease caused by the bacterium Mycobacterium tuberculosis The bacteria can attack any part of the body but usually attacks the lungs Tuberculosis of the lung is spread through the air by coughing talking or sneezing The risk of infection is related to the proximity and the duration of exposure to the source patient Decreased ventilation in crowded and confined environments is often a contributing factor In the United States more than 16000 cases were reported in 2000 and a significant outbreak was reported in Seattle in 2003 People with TB can be treated and cured if they seek medical help but the course of treatment is long However multi drug resistant tuberculosis has emerged as a crucial threat due to inconsistent or partial treatment TB affects around 8 million people and kills about 2 million people each year Today HIV is accelerating the spread of tuberculosis because HIV weakens the immune system Clinical Features In the lungs M tubercqusis may produce a cough that lasts longer than two weeks pain in the chest fatigue fever loss of appetite and coughing up blood or sputum Tuberculosis is a contagious disease spreading through the air like the common cold Only people who are sick with pulmonary TB are infectious When infectious people cough sneeze talk or spit they propel TB bacilli into the air A person needs only to inhale a small number of these to be infected Other human pathogens belonging to the Mycobacterium genus include Mycobacterium avium complex comprising M avium and Mintrace11u1are which causes a TB like disease especially prevalent in AIDS patients and Mycobacterium Ieprae the causative agent of leprosy Mycobacterium tuberculosis is a fairly large nonmotile rodshaped bacterium distantly related to the Actinomycetes Many non pathogenic mycobacteria are components of the normal ora of humans found most often in dry and oily locales The rods are 2 4 um in length and 02 05 um in width Mycobacterium mbercqusis is an obligate aerobe For this reason in the classic case of tuberculosis the M tubercqusis complexes are always found in the well aerated upper lobes of the lungs The bacterium is a facultative intracellular parasite usually of macrophages and has a slow generation time 15 20 hours a physiological characteristic that may contribute to its virulence 11 10 Two media are used to grow M tuberculosis Middlebrook39s medium which is an agar based medium and LowensteinJensen medium which is an egg based medium M tuberculosis colonies are small and buff colored when grown on either medium Both types of media contain inhibitors to keep contaminants from out growing Mycobacteria It takes 46 weeks to get visual colonies on either type of media The cell wall structure of Mycobacterium tuberculosis deserves special attention because it is unique among procaryotes and it is a major determinant of virulence for the bacterium The cell wall complex contains peptidoglycan but otherwise it is composed of complex lipids Over 60 of the mycobacterial cell wall is lipid The lipid fraction of M tuberculosis consists of Mycolic acids and Cord factor Mycolic acids are unique alpha branched lipids found in cell walls of Mycobacterium They make up 50 of the dry weight of the mycobacterial cell envelope Mycolic acids are strong hydrophobic molecules that form a lipid shell around the organism and affect permeability properties at the cell surface Mycolic acids are thought to be a significant determinant of virulence in M tuberculosis Cord Factor Cord factor is toxic to mammalian cells and is also an inhibitor of PMN migration Cord factor is most abundantly produced in virulent strains of Mtuberculosis SPUTUM DIGESTION The majority of clinical specimens submitted to the tuberculosis culture laboratory are contaminated to varying degrees by more rapidly growing normal flora organisms These would rapidly quot 9 overgrow the entire surface of the medium and digest it before 1 the tubercle bacilli start to grow Most specimens must therefore be subjected to a harsh digestion and decontamination procedure that liquefies the organic debris and eliminates the unwanted normal flora EXERCISE 33 AQQID FAST STAIN ACIDFAST STAIN Acid fast staining is based on the high content of mycolic acids present in acid fast organisms that are resistant to staining Unlike Gram positive organisms it is best to use older organisms for this staining technique because younger Mycobacterium may not be as acid fast as older ones since they have not accumulated as much mycolic acid To stain the organisms a strongly reactive dye such as Carbol fuchsin is used It penetrates the cell combines with any mycolic acids present in the cell and binds tightly to them Cells containing high concentrations of mycolic acids cannot be decolorized but non acid fast cells are able to be decolorized Theses non acid fast cells can be counter stained so they can be seen as well Color plate 1 12 11 11 WE Mycobacterium phIei and Staphylococcus epidermidis will be provided as a simulated sputum sample You will Acid fast stain the sample and determine if the patient is presumptive positive for tuberculosis PROCEDURE Step 1 Clean and label the bottom of a microscope silde Step 2 Using your inoculating loop aseptically place a few drops of Simulated Sputum in the center of the slide and smear to about the size of a quarter Air Dry Heat Fix Step 3 Place the fixed slides on a staining rack with the smeared side facing up cover the entire slide with carbol fuchsin Step 4 Heat the slides from above and below with your Bunsen burner isteam gently for 3 min The instructor will demonstrate this technique Step 5 Allow the slide to cool Rinse gently with tap water to remove excess stain Step 6 Decolourize using Acid Alcohol 3 HCl in 95 Ethanol 20 sec Rinse gently with water Step 7 Counterstain with methylene blue for 30 seconds Rinse gently blot dry Step 8 Observe slide with Oil Immersion for Acid Fast bacillus and Nonacid Fast cells Record observations below outlining cell groupings color and staining reaction ACID FAST STAIN DIAGRAM 1000X 11 12 W AEROBIC ANAEROBIC FACULTATIVE ANAEROBIC REDUCED OXIDIZED TUBERCLE BACILLI ACID FAST BACILLI 1 How are we able to successfully transfer cell cultures of CIostridium in the laboratory at our bench completely exposed to oxygen 2 Describe the most common anaerobic pathogen we nd in the clinical laboratory and the type of disease it causes 3 Describe two methods for creating an anaerobic enVironment 4 If you wanted to culture a wound specimen and and couldn t find a Gas Pac Jar would a candle jar serve as a suitable substitute explain 5 How do endospores have medical importance 11 13 6 Can we observe the difference between Gram stained smears of C botuIinum and C tetam explain 7 What is Pseudomembranous colitis What Clostridium species is responsible How do we contract the disease 8 Name three types of Botulism 9 If a patient on a surgical unit develops gas gangrene what hospital precautions if any should be taken why 10 Why do we digest sputum samples that are to be cultured for Mycobacterium 11 Why are tubercle bacilli dif cult to destroy 12 What special precautions are necessary for collecting and handling specimens from tuberculosis patients 13 Is the presence of acid fast bacilli in a sputum sufficient eVidence of tuberculosis why 14 In the United States an acid fast isolate from an AIDS patient is most likely to be which mycobacterial species 11 14