- Site: GIT (Gastrointestinal Track)
- Agent: VIBRIO CHOLERA
- They produce smooth, convex, round, colonies which appear opaque and granular in transmitted light.
- They can grow on many kinds of media including enriching media contains bile salt and asparagine.
- They particularly grow on TCB agar (Thiosulfate Citrate Bile Salt agar) and produce yellow colonies.
- They are readily killed by acid and optimum pH for growth is 8.5-9.5.
- Vibrio Cholera contains two types of antigen flagellar (H) and somatic (O).
- Vibrio Cholera contains two types of antigen flagellar (H) and somatic (O).
- All Vibrios shared a single heat labile H antigen.
- The O antigen is composed of heat stable polysaccharides and are classified into 6 serogroups and are further classified into 60 serotypes on the basis of O antigen.
- One serotype of Vibrio Cholera bacilli is responsible for epidemic cholera and is subdivided into two types.
(1) Classical (2) El Tor
- El Tor types Vibrios were different from the classical types in their ability to cause lysis of goa or sheep erythrocyte in a test known as Grieg Test.
- Each of the two biotypes of 01 serotypes of Vibrio is comprised of two or three antigenic factor A, B, and C
- Factor A and B are found in serotype Ogawa, A and C in serotype Inaba and A, B and C in serotype Hikojima.
- Good water supply. Proper treatment of water should be there before supply to the town.
- Proper treatment of sewerage system.
- Personal hygiene and proper sanitation.
|1. Normal||0-trace||0-2||0-2||Occasional (Hyaline)||–|
|2. Acute glomerulonephritis||1-2+||Numerous;dysmorphic||0-few||Red cell, granular||Smoky urine or hematuria|
|3. Nephrotic syndrome||> 4+||0-few||0-few||Fatty, hyaline, Waxy, epithelial||Oval fat bodies, lipiduria|
|4. Acute pyelonephritis||0-1+||0-few||Numerous||WBC, granular||WBC clumps, bacteria, nitrite test|
|HPF: High power field; LPF: Low power field; RBCs: Red blood cells; WBCs: White blood cells.|
- Clumps of pus cells or pus cells >10/HPF
- Positive nitrite test
- Microscopic examination: In a wet preparation, presence of bacteria should be reported only when urine is fresh. Bacteria occur in combination with pus cells. Gram’s-stained smear of uncentrifuged urine showing 1 or more bacteria per oil-immersion field suggests presence of > 105 bacterial colony forming units/ml of urine. If many squamous cells are present, then urine is probably contaminated with vaginal flora. Also, presence of only bacteria without pus cells indicates contamination with vaginal or skin flora.
- Chemical or reagent strip tests for significant bacteriuria: These are given earlier.
- Culture: On culture, a colony count of >105/ml is strongly suggestive of urinary tract infection, even in asymptomatic females. Positive culture is followed by sensitivity test. Most infections are due to Gram-negative enteric bacteria, particularly Escherichia coli.
- Noncellular: Hyaline, granular, waxy, fatty
- Cellular: Red blood cell, white blood cell, renal tubular epithelial cell.
- Uric acid crystals: These are variable in shape (diamond, rosette, plates), and yellow or red-brown in color (due to urinary pigment). They are soluble in alkali, and insoluble in acid. Increased numbers are found in gout and leukemia. Flat hexagonal uric acid crystals may be mistaken for cysteine crystals that also form in acid urine.
- Calcium oxalate crystals: These are colorless, refractile, and envelope-shaped. Sometimes dumbbell-shaped or peanut-like forms are seen. They are soluble in dilute hydrochloric acid. Ingestion of certain foods like tomatoes, spinach, cabbage, asparagus, and rhubarb causes increase in their numbers. Their increased number in fresh urine (oxaluria) may also suggest oxalate stones. A large number are seen in ethylene glycol poisoning.
- Amorphous urates: These are urate salts of potassium, magnesium, or calcium in acid urine. They are usually yellow, fine granules in compact masses. They are soluble in alkali or saline at 60°C.
- Calcium carbonate crystals: These are small, colorless, and grouped in pairs. They are soluble in acetic acid and give off bubbles of gas when they dissolve.
- Phosphates: Phosphates may occur as crystals (triple phosphates, calcium hydrogen phosphate), or as amorphous deposits.
• Phosphate crystals
Triple phosphates (ammonium magnesium phosphate): They are colorless, shiny, 3-6 sided prisms with oblique surfaces at the ends (“coffinlids”), or may have a feathery fern-like appearance.
Calcium hydrogen phosphate (stellar phosphate): These are colorless, and of variable shape (starshaped, plates or prisms).
• Amorphous phosphates: These occur as colorless small granules, often dispersed.
All phosphates are soluble in dilute acetic acid.
- Ammonium urate crystals: These occur as cactus-like (covered with spines) and called as ‘thornapple’ crystals. They are yellow-brown and soluble in acetic acid at 60°C.
- Cysteine crystals: These are colorless, clear, hexagonal (having 6 sides), very refractile plates in acid urine. They often occur in layers. They are soluble in 30% hydrochloric acid. They are seen in cysteinuria, an inborn error of metabolism. Cysteine crystals are often associated with formation of cysteine stones.
- Cholesterol crystals: These are colorless, refractile, flat rectangular plates with notched (missing) corners, and appear stacked in a stair-step arrangement. They are soluble in ether, chloroform, or alcohol. They are seen in lipiduria e.g. nephrotic syndrome and hypercholesterolemia. They can be positively identified by polarizing microscope.
- Bilirubin crystals: These are small (5 μ), brown crystals of variable shape (square, bead-like, or fine needles). Their presence can be confirmed by doing reagent strip or chemical test for bilirubin. These crystals are soluble in strong acid or alkali. They are seen in severe obstructive liver disease.
- Leucine crystals: These are refractile, yellow or brown, spheres with radial or concentric striations. They are soluble in alkali. They are usually found in urine along with tyrosine in severe liver disease (cirrhosis).
- Tyrosine crystals: They appear as clusters of fine, delicate, colorless or yellow needles and are seen in liver disease and tyrosinemia (an inborn error of metabolism). They dissolve in alkali.
- Sulfonamide crystals: They are variably shaped crystals, but usually appear as sheaves of needles. They occur following sulfonamide therapy. They are soluble in acetone.
- Whenever the microscope is not in use, turn off the illuminator. This will greatly extend the life of the bulb, as well as keep the temperature down during extended periods of laboratory work.
- When cleaning the microscope, use distilled water or lens cleaner. Avoid using other chemicals or solvents, as they may be corrosive to the rubber or lens mounts.
- After using immersion oil, clean off any residue immediately. Avoid rotating the 40× objective through immersion oil. If this should occur, immediately clean the 40× objective with lens cleaner before the oil has a chance to dry.
- Do not be afraid to use many sheets of lens tissue when cleaning. Use a fresh piece (or a clean area of the same piece) when moving to a different part of the microscope. This avoids tracking dirt/oil/residue to other areas of the microscope.
- Store the microscope safely with the stage lowered and the smallest objective in position (4× or 10×). This placement allows for the greatest distance between the stage and the objective. If the microscope is bumped, the likelihood of an objective becoming damaged by the stage surface will be greatly minimized.
- Red cells: Morphology, immature forms, inclusion bodies, arrangement of cells.
- White cells: Differential count, abnormal or immature forms.
- Platelets: Adequacy, abnormal forms.
- Parasites: Malaria, filaria.
- Acute bacterial infections: Abscess, pneumonia, meningitis, septicemia, acute rheumatic fever, urinary tract infection.
- Tissue necrosis: Burns, injury, myocardial infarction.
- Acute blood loss
- Acute hemorrhage
- Myeloproliferative disorders
- Metabolic disorders: Uremia, acidosis, gout
- Malignant tumors
- Physiologic causes: Exercise, labor, pregnancy, emotional stress.
- Severe bacterial infections, e.g. septicemia, pneumonia
- Severe hemorrhage
- Severe acute hemolysis
- Carcinoma metastatic to bone marrow Leukemoid reaction should be differentiated from chronic myeloid leukemia (Table 801.1).
(a) Bacterial: typhoid, paratyphoid, miliary tuberculosis, septicemia
(b) Viral: influenza, measles, rubella, infectious mononucleosis, infective hepatitis.
(c) Protozoal: malaria, kala azar
(d) Overwhelming infection by any organism
- Hematologic disorders: megaloblastic anemia, aplastic anemia, aleukemic leukemia, myelophthisis.
(a) Idiosyncratic action: Analgesics, antibiotics, sulfonamides, phenothiazines, antithyroid drugs, anticonvulsants.
(b) Dose-related: Anticancer drugs
- Ionizing radiation
- Congenital disorders: Kostman's syndrome, cyclic neutropenia, reticular dysgenesis.
- Neonatal isoimmune neutropaenia
- Systemic lupus erythematosus
- Felty's syndrome
- Allergic diseases: Bronchial asthma, rhinitis, urticaria, drugs.
- Skin diseases: Eczema, pemphigus, dermatitis herpetiformis.
- Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis.
- Hematologic disorders: Chronic Myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma.
- Carcinoma with necrosis.
- Radiation therapy.
- Lung diseases: Loeffler's syndrome, tropical eosinophilia
- Hypereosinophilic syndrome.
- Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar.
- Recovery from neutropenia.
- Autoimmune disorders.
- Hematologic diseases: Myeloproliferative disorders, monocytic leukemia, Hodgkin's disease.
- Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis.
(a) Viral: Acute infectious lymphocytosis, infective hepatitis, cytomegalovirus, mumps, rubella, varicella
(b) Bacterial: Pertussis, tuberculosis
(c) Protozoal: Toxoplasmosis
- Hematological disorders: Acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma.
- Other: Serum sickness, post-vaccination, drug reactions.
- Polymorphonuclear neutrophil: Neutrophil measures 14-15 μm in size. Its cytoplasm is colorless or lightly eosinophilic and contains multiple, small, fine, mauve granules. Nucleus has 2-5 lobes that are connected by fine chromatin strands. Nuclear chromatin is condensed and stains deep purple in color. A segmented neutrophil has at least 2 lobes connected by a chromatin strand. A band neutrophil shows non-segmented U-shaped nucleus of even width. Normally band neutrophils comprise less than 3% of all leukocytes. Majority of neutrophils have 3 lobes, while less than 5% have 5 lobes. In females, 2-3% of neutrophils show a small projection (called drumstick) on the nuclear lobe. It represents one inactivated X chromosome.
- Eosinophil: Eosinophils are slightly larger than neutrophils (15-16 μm). The nucleus is often bilobed and the cytoplasm is packed with numerous, large, bright orange-red granules. On blood smears, some of the eosinophils are often ruptured.
- Basophils: Basophils are seen rarely on normal smears. They are small (9-12 μm), round to oval cells, which contain very large, coarse, deep purple granules. It is difficult to make out the nucleus since granules cover it.
- Monocytes: Monocyte is the largest of the leukocytes (15-20 μm). It is irregular in shape, with oval or clefted (kidney-shaped) nucleus and fine, delicate chromatin. Cytoplasm is abundant, bluegray with ground glass appearance and often contains fine azurophil granules and vacuoles. After migration to the tissues from blood, they are called as macrophages.
- Lymphocytes: On peripheral blood smear, two types of lymphocytes are distinguished: small and large. The majority of lymphocytes are small (7-8 μm). These cells have a high nuclearcytoplasmic ratio with a thin rim of deep blue cytoplasm. The nucleus is round or slightly clefted with coarsely clumped chromatin. Large lymphocytes (10-15 μm) have a more abundant, pale blue cytoplasm, which may contain a few azurophil granules. Nucleus is oval or round and often placed on one side of the cell.
- Toxic granules: These are darkly staining, bluepurple, coarse granules in the cytoplasm of neutrophils. They are commonly seen in severe bacterial infections.
- Döhle inclusion bodies: These are small, oval, pale blue cytoplasmic inclusions in the periphery of neutrophils. They represent remnants of ribosomes and rough endoplasmic reticulum. They are often associated with toxic granules and are seen in bacterial infections.
- Cytoplasmic vacuoles: Vacuoles in neutrophils are indicative of phagocytosis and are seen in bacterial infections.
- Shift to left of neutrophils: This refers to presence of immature cells of neutrophil series (band forms and metamyelocytes) in peripheral blood and occurs in infections and inflammatory disorders.
- Hypersegmented neutrophils: Hypersegmentation of neutrophils is said to be present when >5% of neutrophils have 5 or more lobes. They are large in size and are also called as macropolycytes. They are seen in folate or vitamin B12 deficiency and represent one of the earliest signs.
- Pelger-Huet cells: In Pelger-Huet anomaly (a benign autosomal dominant condition), there is failure of nuclear segmentation of granulocytes so that nuclei are rod-like, round, or have two segments. Such granulocytes are also observed in myeloproliferative disorders (pseudo-Pelger-Huet cells).
- Atypical lymphocytes: These are seen in viral infections, especially infectious mononucleosis. Atypical lymphocytes are large, irregularly shaped lymphocytes with abundant cytoplasm and irregular nuclei. Cytoplasm shows deep basophilia at the edges and scalloping of borders. Nuclear chromatin is less dense and occasional nucleolus may be present.
- Blast cells: These are most premature of the leukocytes. They are large (15-25 μm), round to oval cells, with high nuclear cytoplasmic ratio. Nucleus shows one or more nucleoli and nuclear chromatin is immature. These cells are seen in severe infections, infiltrative disorders, and leukemia. In leukemia and lymphoma, blood smear suggests the diagnosis or differential diagnosis and helps in ordering further tests (see Figure 800.2 and Box 800.1).
Parts and functions of a compound microscope
(A) Arm: Used to carry the microscope.
Kohler illumination is a method of adjusting a microscope in order to provide optimal illumination by focusing the light on the specimen. When a microscope is in Kohler, specimens will appear clearer, and in more detail.
Process of setting Kohler
- Specimen slide (will need tofocus under 10× power)
- Compound microscope.
- Mount the specimen slide onthe stage and focus under 10×.
- Close the iris diaphragm completely.
- If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
- Using the condenser adjustment knobs, raise or lower the condenser until the edges of the field becomes sharp (see Figure 797.1 and Figure 797.2).
- Open the iris diaphragm until the entire field is illuminated.
- During regular microscope maintenance
- After the microscope is moved/transported
- Whenever you suspect objects do not appear as sharp as they could be.
The theory of biogenesis states that living things can only arise from living things and cannot be spontaneously generated. Learn more about this popular microbiology theory to better understand what it means.
The Spontaneous generation hypothesis proposed by scientists to explain the origin of the “animalcules" observed by Antoni van Leeuwenhoek in his magnifying lenses had received wide acceptance all over Europe from Antoni’s time until the time of Louis Pasteur. Erroneous experimental set up, results, and conclusions of some scientists had supported and strengthened the hypothesis.
For example, the Englishman John Needham claimed that vital life is needed for the spontaneous generation of microbes. He added that the reason why no living organisms emerged from heated and sealed solutions in containers is that the “vital life" was destroyed by the heat and new “vital life" was not supplied to the solutions because they cannot enter the sealed containers.
Fortunately, there were scientists skeptical about the hypothesis, so they designed their own experimental set up and from the results they gathered, they drew the most feasible explanation on the origin of the “animalcules".Among the scientists was the Italian Lazzaro Spallanzani who opposed Needham’s idea of the “vital life".
Proponents and opponents of spontaneous generation hypothesis debated a lot starting from the time Leeuwenhoek presented his discoveries (1670s) to the public until the time of Rudolf Virchow, who in 1858 challenged the spontaneous generation with his concept and definition of biogenesis.
This concept claims that living cells can arise only from preexisting living cells. Virchow defended this concept to the scientific community but he did not come up with a convincing experiment to back up his idea. In 1861, the French scientist Louis Pasteur resolved the issue on the origin of microbes (“animalcules") through a series of ingenious and persuasive experiments.
Pasteur showed that microorganisms exist in the air and can contaminate sterile solutions, but he emphasized that air itself does not produce microbes. He filled a number of short-necked flasks with beef broth and then boiled their contents. He immediately sealed the mouths of some of the flasks while he left the others open and allowed to cool.
After few days, the contents of the unsealed flasks were found to be contaminated with microorganisms. No evidences of growing microorganisms were found on the sealed flasks. Pasteur concluded that the microorganisms in the air were responsible for contaminating non-living matter like the broths in John Needham’s flask.
Pasteur performed another experiment but this time he put beef broth in open-ended long-necked flasks. He bent the necks of the flasks into S-shaped curves and boiled the contents of the flasks. Amazingly, the contents of the flasks were not contaminated even after several months.
The unique S-shaped design of Pasteur’s flasks allowed air to pass but trap microorganisms that may contaminate the broths. Do you know that some of the original vessels used by Pasteur in his experiments are still displayed in the Pasteur Institute, Paris today? A few of the flasks contain broths that remain uncontaminated for more than 100 years!
Pasteur demonstrated the presence of microbes in non-living materials whether they are solid, liquid, or air. In addition, he laid the foundation of aseptic techniques, techniques that prevent contamination by unwanted microbes.
These techniques are based on Pasteur’s idea that microbes can be killed by heat and that procedures can be designed to inhibit the access of airborne microbes to nutrient environment. Application of aseptic techniques is now the standard practice in medical and laboratory procedures.
Disproving the idea that microorganisms spontaneously generated from non-living matter through mystical forces is one of the greatest contributions of Pasteur in science. He provided the evidence that any appearance of “spontaneous" life in nonliving solutions can be attributed to microbes that already exist in the air or in the fluids themselves.