Bacteria Information and Courses from MediaLab, Inc.

Spinal fluid samples are either clear or turbid. Some sources use the following rating system for turbid CSF specimens: 0 = crystal clear fluid 1+ = faintly cloudy, smoky, or hazy 2+ = turbidity clearly visible but newsprint read easily through tube 3+ = newsprint not easily read through tube 4+ = newsprint cannot be seen through the tubeTurbidity may be caused by leukocytes, erythrocytes, fungi, bacteria, amoebae, contrast media, or aspiration of epidural fat during puncture.200 leukocytes/mm3 will cause slight turbidity (1+); increased numbers of WBCs will cause increased turbidity. At least 400 erythrocytes/mm3 are needed to produce 1+ turbidity.Occasionally CSF will have an oily appearance due to the presence of substances remaining in the CSF after radiologic (x-ray) procedures have been performed.

Reasons for acidic urine pH include: a high-meat diet, respiratory/metabolic acidosis, and hypochloridemia. A urine with a high concentration of glucose may also have a lower pH. An alkaline pH may be the result of a vegetarian diet, respiratory/metabolic alkalosis, or a bacterial infection caused by urease-producing bacteria. Urine that contains bacteria can become more alkaline if the specimen remains at room temperature for an extended period of time.

The nitrites portion of the reagent strip provides a rapid screening test for the presence of gram-negative bacteria that are often responsible for urinary tract infections. Although urine cultures are still needed to confirm the diagnosis and monitor any urinary tract or kidney infection, the need for a culture may not be obvious because in some cases of early bladder infection, the symptoms may be vague or the patient may be asymptomatic. Diagnosis and treatment of cystitis (bladder infection) is important because if left untreated it may result in kidney damage, impairment of renal function, hypertension and/or septicemia.

This test is sensitive to 0.06-0.1 mg/dL nitrite ion in urines with a low specific gravity and ascorbic acid concentrations of less than 25 mg/dL. Pink spots or pink edges should not be interpreted as a positive result because some medications can color urine red or turn red in an acid environment. Any degree of uniform pink color should be considered positive, suggesting the presence of 105 organisms/mL. Detection of low levels of nitrite ion may be enhanced by comparing the activated test strip to a white background. It is important to note that color development is NOT proportional to the number of bacteria present. The test is specific for nitrites and does not react with any other substances normally present in urine. Negative results do not necessarily rule out a urinary tract infection because yeasts or gram-positive bacteria unable to reduce nitrites may be the causative agent.

Early detection of bacteria is important in order to prevent cystitis from developing into inflammation or infection involving the kidney and renal pelvis. The nitrite portion of the test strip can be used to screen individuals who are at risk for developing urinary tract infections, such as diabetics, persons with recurrent infections, or pregnant women. The test is also useful in evaluating the success of antibiotic therapy that is used to treat a bladder infection.

Urobilinogen is a byproduct of hemoglobin breakdown. It is produced in the intestinal tract as a result of the action of bacteria on bilirubin. Almost half of the urobilinogen produced recirculates through the liver and then returns to the intestines through the bile duct. Urobilinogen is then excreted in the feces where it is converted to urobilin. As the urobilinogen circulates in the blood to the liver, a portion of it is diverted to the kidneys and appears as urinary urobilinogen. Up to 1 mg/dL or Ehrlich unit of urobilinogen is present in normal urine. A result of 2.0 mg/dL represents the transition from normal to abnormal and the patient should be evaluated further. It is important to note that the reagent strip cannot determine the absence of urobilinogen.

Prompt testing (within one hour of collection of the urine sample) or refrigeration of the specimen is necessary because the glycolytic enzymes from the cells and bacteria, if present, will decrease the sugar in the urine.

Citron DM. Appelbaum PC.: How far should a clinical laboratory go in identifying anaerobic isolates, and who should pay? Clinical Infectious Diseases. 16 Suppl 4:S435-8, 1993Identification of anaerobic bacteria in specimens from sites of infection due to mixed organisms can be time-consuming and expensive. Laboratories should limit anaerobic workups by testing only those specimens that have been properly collected and transported to the laboratory.Use of selective and differential media for initial processing can provide rapid and relevant information to the clinician. Anaerobes isolated from normally sterile sites and sites of serious infection should always be completely identified. Group-or genus-level identifications may suffice in other instances.The Bacteroides fragilis group of organisms should always be identified because of their virulence and resistance to many antimicrobial agents.Some of the other organisms that warrant identification include Clostridium septicum (associated with gastrointestinal malignancy); Clostridium ramosum, Clostridium innocuum, and Clostridium clostridioforme (which are resistant to antibiotics); Clostridium perfringens (a cause of myonecrosis and gas gangrene,potentially serious infection); anaerobic cocci (which may be resistant to metronidazole and clindamycin); and fusobacteria (which may be virulent and resistant to clindamycin and penicillin).

In follow up to the previous question, the upper image again illustrates the colonies recovered from the blood culture bottle. The colonies are small, transluscent, gray-yellow, and surrounded by a wide zone of beta hemolysis.The size of the colonies compared to the zones of hemolysis suggests a group A streptococcus.The susceptibility to bacitracin (zone of inhibition around the "A" disk)(lower photograph) is virtually diagnostic of a group A streptococcus.The absence of a zone of inhibition around the SXT disk indicates resitance to sulfamethoxazole/ trimethoprim. SXT resistance is also shared by group B streptococci, which are, however, resistant to bacitracin.The resistance to SXT is used for the primary recovery of groups A and B streptococci from specimens with mixed culture. Their resistance allows them to selectively grow out from contaminating bacteria that are inhibited by this antibiotic.

Rouquette C. Berche P. The pathogenesis of infection by Listeria monocytogenes Microbiologia. 12:245-58, 1996 Listeria monocytogenes is a Gram-positive bacterium responsible for severe infections in human and a large variety of animal species. It is a facultative intracellular pathogen which invades macrophages and most tissue cells of infected hosts where it can proliferate. The molecular basis of this intracellular parasitism has been to a large extent elucidated. The virulence factors, including internalin, listeriolysin O, phospholipases and a bacterial surface protein, ActA, are encoded by chromosomal genes organized in operons. Following internalisation into host cells, the bacteria escape from the phagosomal compartment and enter the cytoplasm. They then spread from cell to cell by a process involving actin polymerisation. In infected hosts, the bacteria cross the intestinal wall at Peyer's patches to invade the mesenteric lymph nodes and the blood. The main target organ is the liver, where the bacteria multiply inside hepatocytes. Early recruitment of polymorphonuclear cells lead to hepatocyte lysis, and thereby bacterial release This causes prolonged septicaemia, particularly in immunocompromised hosts, thus exposing the placenta and brain to infection. The prognosis of listeriosis depends on the severity of meningoencephalitis, due to the elective location of foci of infection in the brain stem (rhombencephalitis). Despite bactericidal antibiotic therapy, the overall mortality is still high (25 to 30%).

Biological agents are organisms or toxins that can kill or incapacitate people, live stock, and crops. The three basic groups of biological agents that would likely be used as weapons are bacteria, viruses, and toxins. Biological agents can be dispersed as aerosols or airborne particles.

Examining the biopsy allows the structure of the marrow to be viewed as it exists in the body. It provides essential diagnostic information in conditions that disrupt the normal architecture, such as metastatic carcinoma, myelofibrosis, Hodgkin's lymphoma and granuloma. A biopsy may also be used to evaluate cellularity and identify acid-fast bacteria or fungi in less time than is needed for routine culture methods. One disadvantage of the tissue sections prepared from the biopsy sample is that morphologic detail is lost. For this reason, in many cases imprint slides or smears from the aspirated sample are also examined.

It is possible that since anti-A and anti-B develop so predictably, without a recognizable immunizing event, that they are “naturally” occurring. Their production is thought to be stimulated by bacteria which have been shown to contain substances that are chemically similar to human A and B antigens. (Anti-A and anti-B are absent in germ-free animals.) Except for the rare hh individuals who lack H substance, everyone has some H in their cellular makeup.

Specialists in microbiology perform testing to diagnose and stop the spread of infectious organisms, including bacteria, viruses, and parasites. Specialists should be able to isolate and identify a wide variety of these organisms. Testing procedures include direction examination and antigen detection methods. Specialists in serology and immunology measure antibodies to infectious organisms. Specialists should be familiar with all serology techniques (except those specific to immunohematology). This specialty includes all lab procedures performed in the specialty of histocompatibility. Specialists in hematology must be able to identify and evaluate cells in blood and bone marrow and identify disorders of these cell. Specialists should be familiar with routine and special tests to determine the number, morphology, and function of cells in body fluid.

Monocytes provide defense against mycobacteria, fungi, bacteria, protozoa and viruses. They respond to chemotactic factors, phagocytize and kill the microorganisms.

Follow this order whenever you wash your hands: Remove and dispose of gloves. Wet hands. Use liquid soap (bacteria live in soap dishes). Wash for at least 10 seconds cleaning all surfaces (including under fingernails). Don't touch faucets after washing (Don't recontaminate your hands). Dry with paper towels. Turn off faucets with paper towels.

Are used to collect sterile blood samples from patients who may be septic (have bacteria or other organisms growing in their bloodstream). Different blood culture bottles are used for aerobic, anaerobic, and pediatric collections.

Finally, prep the site with an iodine swab. Start at the center of the site, and move outward in enlarging concentric circles. Do not go over the same area twice.Stop when you have covered an area about 4 inches in diameter. Allow this area to dry for at least one minute so that the iodine has time to kill the skin bacteria.

Clean the site thoroughly with alcohol, then with iodine, to rid the skin of contaminating bacteria.Next, clean the site again with alcohol, and allow it to dry.

Although this smear is the proper thickness, it is not appropriate for examination because the nonbacterial elements are stained blue instead of red.In addition, small flecks of precipitated stain are present.Notice that the precipitated stain is irregular in shape, which helps differentiate the flecks from bacteria.The stain should be filtered before using.

Sometimes bacteria are present within white cells. In this field, there are gram negative bacteria present within a neutrophil.

Epithelial cells are larger than white blood cells and red blood cells, and contain a single nucleus. White blood cells (pus cells) usually show a segmented nucleus. Red blood cells are 1/2 to 2/3 as large as white blood cells, contain no nucleus, and are gram negative.Hyphae are gram positive tubular filamentous fungal elements which may show branching or intertwining. Yeast cells are round to oval, often budding, gram positive fungal elements, about the same size as RBCs. They are generally much larger than bacteria.

Epithelial cells in large numbers within sputum smears means that the specimen is predominantly oral saliva, rather than true sputum from the lung. Epithelial cells in urine smears indicate that the sample has been contaminated by organisms found on the vulva or distal urethra. Bacteria found near or on epithelial cells are usually normal contaminating bacterial flora.White blood cells indicate inflammation and possible infection. The direct smear examination should focus within and around these cells.Red blood cells in a direct smear are not usually significant.Yeast may be present as normal flora in upper respiratory tract or genital tract. They may be significant if they predominate, or if budding yeast forms are seen.Hyphae are more likely to indicate the presence of fungal infection, but this determination requires correlation with clinical findings.Bacteria found in spinal fluid, blood, tissue and specimens from other sterile sites are always significant.Body fluids which are normally sterile must be examined carefully. If only one organism per oil immersion field is identified, then there are about 105 organisms per mL present in the sample! Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm.

Notice that Gram positive and Gram negative organisms can be either cocci (round) or bacilli (rod-shaped). A mixture Gram positive and Gram negative bacteria may be present in the same field.

Clumps or clusters of Gram positive bacteria are evident in this slide.

This field shows examples of Gram negative bacteria appearing in pairs.

The Gram stain provides a fundamental differentiation between types of bacteria. Gram positive bacteria retain the primary stain and are deep violet in color. Gram negative organisms are those that decolorize during the staining process and, after counterstaining, are pink in color. Bacteria have two basic shapes:cocci (round)bacilli (rod-shape)

Examples of Gram positive bacteria appearing in tetrads are seen in this slide. Sometimes bacteria in tetrad may not be as clear cut as this drawing indicates, and appear as clusters. N. gonorrhea colonies may appear as tetrads especially if the smear is made from very young colonies.

Examination of a fresh urine specimen provides the best results. If a specimen cannot be examined immediately, it may be refrigerated for up to 12 hours. Refrigeration prevents decomposition of casts, cells and the overgrowth of bacteria. The urine sediment elements begin to lyse after 1-3 hours at room temperature. Although the most commonly received urine specimen is the random urine collection, the specimen of choice for urinalysis is the first morning urine. The first morning urine is more concentrated and allows for the detection of substances which may not be present in a more dilute random sample. Once the physical and chemical characteristics of the urine have been determined, the microscopic exam is performed on the sediment.

Bacteria may also be present, especially during a urinary tract infection. This view shows bacteria as solid gray rods or cocci. Since bacteria may also be a contaminant in specimens remaining at room temperature, or due to an unclean catch, caution must be observed in reporting bacteria. If 20 organisms per hpf are seen, the bacteria are considered to be clinically significant.

The results are abnormal. The presence of glucose is not a normal finding. However, the two glucose methods correlate well with each other.The specific gravity does not correlate well with the glucose. A large amount of glucose should elevate the urine specific gravity. The specific gravity result should therefore be rechecked before reporting. The presence of 3+ bacteria, does not correlate well with scant white cells and lack of turbidity. The technologist should question whether the specimen was held at room temperature for a protracted period prior to examination.

A mixture of bacteria and amorphous material is seen in this field. In order to determine the approximate number of bacteria, the amorphous material can be eliminated.

The natural history of TB infection is usually followed by an immune response and latency after exposure. In about 5-10% of cases, the latent period progresses to an active infection.The organism that causes TB infection is Mycobacterium tuberculosis. This organism is pictured in the photograph to the right as observed when stained with acridine orange stain. Infection occurs when a susceptible person inhales droplet nuclei containing Mycobacterium tuberculosis and the organism reaches the alveoli of the lungs.About 2-12 weeks after infection, the immune system limits multiplication of additional bacteria and the immunological test becomes positive.Latent tuberculosis infection (LTBI) is the stage when the viable organism remains in the body, and the patient has no symptoms and is non-infectious.Most infected persons do not experience clinical illness and are noninfectious. About 5-10% of persons infected with Mycobacterium tuberculosis who are not treated will develop TB during their lifetime. The risk for progression is highest during the first several years after infection.TB infects the lungs most often; however, it can infect almost any organ in the body, including bones and joints.

Many variations in morphology may be seen when examining Wright's stained peripheral blood smears. One method of classifying these variations in white cell morphology is based on the way the body responds to a stimulus, deficiency, or the presence of an inherited defect. This classification falls into three groups:Pathological: Cells may show abnormalities in appearance and/or function. The body is responding abnormally to a stimulus or inherited defect, resulting in physiological impairment in the patient. Nonpathological: Cells may show variation in morphology but their function is normal. Their presence does not cause physiological impairment. Reactive: Cells show variation in morphology but are functioning normally in response to a specific stimulus, such as a virus or bacteria. There is a disease process in progress to which the cells are responding. Although the morphology has varied from normal and their presence is significant, the body is responding normally to a stimulus.

Unusually dark staining Dohle bodies are seen in the cytoplasm of this cell, along with bacteria, an infrequent finding. The bacteria are round and stain almost black. The Dohle bodies are patches of dark blue stained material.

Chediak-Higashi syndrome is a rare autosomal recessive disorder. It results from a mutation of the gene LYST which encodes a protein with multiple phosphorylation sites. This defect causes a cellular abnormality involving the fusion of cytoplasmic granules. Early in neutrophil maturation normal azurophilic granules form, but they fuse together to form megagranules. Later during the myelocyte stage, normal specific granules form. The mature neutrophils contain both normal specific granules and abnormal azurophilic granules. These large abnormal granules can be seen in the cytoplasm of neutrophils, eosinophils, basophils, monocytes and lymphocytes. These abnormal granules are able to kill bacteria in neutrophils and monocytes; however, the process is much less effective than in normal cells in part, because these neutrophils have impaired locomotion. For these reasons, individuals with Chediak-Higashi have recurrent infections. An accelerated lymphoma-like phase occurs, with lymphadenopathy, hepatosplenomegaly, and pancytopenia. Death often occurs at an early age.

The cytoplasm of eosinophils is evenly filled by numerous orange-red granules of uniform size. They do not overlie the nucleus.The eosinophil granules contain numerous enzymes including peroxidase, phospholipase D, catalase, acid phosphatase, and vitamin B12-binding proteins.Their ability to kill bacteria is less than that of neutrophils. Their main purpose is to counteract parasitic infections and to participate in immune allergic reactions.They may also be increased in a variety of nonimmunologic inflammatory responses from bacteria and fungi causing chronic infections. Malignancies, collagen vascular diseases, and myeloproliferative disorders may also may be settings for prominent eosinophils.