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Biological Oxygen Demand (BOD)

It is also known as Biochemical Oxygen Demand (BOD). It is defined as the amount of oxygen consumed during the process of degradation and eventual stabilization of unstable organic substances by the biochemical activities of aerobic and other microbes. This degradation of the chemical complex is a desirable process and the final product is called stabilized wastewater. The aerobic bacteria consume oxygen during the process of oxidization of the organic and other oxidizable inorganic substances. The immensity of biochemical degradation depends on the population of bacteria. An actively growing population of bacteria will consume more oxygen to quickly decompose unstable complexes. Biological/Biochemical Oxygen Demand (BOD) is reduced with the decrease in the quantity of these complexes in the wastewater. Therefore, it can be surmised that BOD is directly proportional to the level of degradable chemical complexes; high concentration of chemical substances will result in the high BOD.

The BOD is a very useful measure of the efficiency of methods of wastewater treatment. A method in which amount of BOD reduced quickly is considered as most effective and efficient method. Therefore, exactly stabilized effluent, when discharge in the body of water, does not cause reduction of oxygen in the water.

Wastewater Disposal Methods

It is a well-known fact that the wastewater should be treated properly and effectively before its disposal into receiving water bodies. Disposal of wastewater may be accomplished with or without treatment.

Disposal of Wastewater by Treatment Methods

There are different methods available for the removal of microorganisms and stabilized the putrescible organic and inorganic chemicals in the wastewater. These methods are known as wastewater treatment methods. It is a very interesting fact that usually microorganisms are used to reduce the large burden of wastewater, which is organic matter. With few exceptions, wastewater treatment plants are integrated with physical, chemical and microbiological methods to concern with the different problems related to wastewater.

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According to distinct types of treatment, they are divided into four types. Each type of treatment process has a special purpose, targeting the removal of all sources of materials and reducing the burden of microorganisms from the wastewater.

Primary Treatment of Wastewater

This process is mainly designed to remove the total solids from the wastewater by sedimentation and render it adequately free from pathogenic bacteria by chlorination. Initially, large objects are removed by bar screens from the wastewater flow. It removes a significant amount of particulate matter. The collected objects are then put into the grinder and released back into the wastewater flow.

The wastewater is then allowed to flow to a series of large primary settling compartments in which most of the organic matters and dense inorganic particles such as grit and sands are removed. Usually, there are two types of settling compartment, (a) grit compartment and (b) sedimentation tank or quiescent settling compartment. In grit compartment, wastewater flows very slow which permits large and heavy particulate matter to settle out. In the next step, the municipal and industrial wastes (particulate organic matters) in wastewater are removed in the sedimentation tank. In sedimentation tank, wastewater is allowed to stay for 1 to 3 hours during which most of the suspended organic matter settles out. The sedimented material is in the form of a semi-solid mass called sludge. The efficiency of sludge formation can be increased by the addition of various chemicals to coagulate the suspended particles which enhanced the sedimentation rate. The sludge is not allowed to remain in the bottom of sedimentation tank for a long period because of anaerobic bacteria produce gases during metabolism that tend to resuspend the settled material and increased the odor. Therefore, the sedimentation tank is equipped with scrapper mechanisms that occasionally removes the bottom sludge to a collection hopper. The underflow sludge becomes a waste product of the process. The remaining liquid portion of the wastewater which leaves the tank is called effluent.

 

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There are certain bacteria which cannot be stained by Gram's method. In 1882, Paul Ehrlich developed a method of staining such type of bacteria. This method was named, and still known as acid-fast staining and the bacteria were named as acid-fast bacteria. In the same year, Ehrlich's method was improved by Zehil and Neelsen. Nowadays, Ziehl-Neelsen method is believed as most important differential staining procedure used for the identification of acid-fast species of Mycobacterium, Actinomyces, and Nocardia. There are many acid-fast bacteria which are pathogenic, such as M. tuberculosis (tuberculosis), A. israelii (actinomycosis), M. leprae (leprosis), and N. asteroides (nocardiosis).

Acid-fast bacteria may be defined as those cells which keep the color of the primary dye (carbol fuchsin) even after the process of decolorization by the acid-alcohol solution. Those bacteria which fail to do so are known as non-acid-fast bacteria.

Acid-fast bacteria are coated with a thick waxy material, mycolic acid, which makes the bacterial cells highly resistant to the penetration of dyes. The penetration of dye is promoted by using heat as mordant. The heat invades the dye through the waxy coat and into the cytoplasm.

PURPOSE

  • Differentiation between acid-fast and non-acid-fast bacteria.
  • Diagnosis of pulmonary tuberculosis from sputum smear. See also: Examination of Sputum

NEEDS

Specimen

Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria grown and isolated in culture.

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Reagents

  1. Carbol fuchsin
  2. Acid-alcohol solution
  3. Methylene blue

Equipment

  1. Bunsen burner
  2. Wire loop
  3. Glass Slide
  4. Spirit lamp
  5. Microscope

PROCEDURE

Acid-Fast staining by Ziehl-Neelsen method

  1. Take a clean glass slide and prepare a thin smear from the specimen using sterile technique. The smear should be extremely thin covering a large area of the slide.
  2. Dry the smear is air and then fix the slide by passing the slide through the flame.
  3. Cover the slide with carbol fuchsin. Keep it for 5 minutes over a spirit lamp with constant heating but not boiling. Do not allow the stain to dry over the slide.
  4. When the slide is cooled, wash it with tap water.
  5. Flood the slide with acid-alcohol and leave it for 3 minutes. Wash the slide with tap water and drain.
  6. Counterstain with methylene blue for 2 minutes.
  7. Wash the slide with tap water and keep it for dry.
  8. Observe the slide under oil immersion objective.

OBSERVATION

Microscopic examination reveals acid-fast tubercle bacilli as short, straight or slightly curved bright red rods. Non-acid-fast cells appear blue.

Acid-Fast Staining by Mobin Method

In 1985, a Pakistani microbiologist, Abdul Mobin Khan developed a method for the staining of acid-fast bacteria. In this method, heating of flooded primary dye on smear is not required. However, initial fixing of the smear over the flame is necessary in order to increase the permeability of the cell wall and promote the newly formulated primary dye to penetrate the cell.

Needs

Specimen

Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria grown and isolated in culture.

Reagents

  1. Mobin stain
  2. 1% H2SO4
  3. Crystal violet

Equipment

  1. Wire loop
  2. Bunsen burner
  3. Glass slides
  4. Microscope

Procedure

  1. Using sterile technique, prepare a thin smear from the specimen covering a large area of the glass slide.
  2. Dry the smear in the air and then fix it by passing the slide 20 times through the flame.
  3. Place the smear on the staining rack and cover it with Mobin stain. Keep it for 10 minutes.
  4. Pour off the stain and wash the slide with tap water.
  5. Decolorize the smear by 1% H2SO4 solution till it is light pink.
  6. Wash the slide with tap water and keep it for dry.
  7. Observer the slide under oil immersion objective.

Observation

Microscopic examination reveals acid-fast tubercle bacilli as short, straight or slightly curved red rods while non-acid-fast bacteria as blue.

In 1883 (originally published in 1884), Dr. Hans Christian Gram (1853-1938) developed a technique for the classification of bacteria into two broad groups, Gram-positive and Gram-negative. It is the most important staining technique for the classification and differentiation of bacteria.

The Gram stain consist of four reagents; crystal violet (use as a primary dye), Gram's iodine (use as a mordant), ethyl alcohol (use as a decolorizer), and safranin (use as a counterstain). The Gram-negative, on the other hand, lose the primary dye (crystal violet) when decolorized and, thus, take the color of counterstain (safranin).

The Gram-reaction rely upon the chemical nature of the bacterial cell wall, especially the lipids which comprise 11-22% in Gram-negative cell wall and 1-4% in the Gram-positive cell wall. In the Gram-negative cell wall, the amount of lipids is very high, when the cell is dissolved in alcohol, it leads to the formation of large pores in the cell wall. The dehydrating result of alcohol cannot fill these pores which cause the liberate of primary stain making the cell colorless. Such cells take the color or counterstain (safranin). On the contrary, the amount of lipid is very low in the Gram-positive cell wall and easily dissolved by in ethyl alcohol, causing the formation of very small pores. These pores are further closed by the dehydrating effect of alcohol which does not permit the primary dye (crystal violet) to leave the cell.

PURPOSE

Identification, differentiation, and classification of the bacteria.

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NEEDS

Specimen

Sputum, body fluid, pus, or swab of cells taken from the location of an infection; a sample of bacteria or fungi grown and isolated in culture.

Reagents

  1. Crystal violet
  2. Gram iodine
  3. Ethyl alcohol (95%)
  4. Safranin
  5. Ceder wood oil

Equipment

  1. Bunsen burner
  2. Wire loop
  3. Glass slides
  4. Microscope

PROCEDURE

  1. Prepare a smear of the specimen, dry in air and then fix it in low flame.
  2. Flood the smear with crystal violet, and keep it for 1 minute. Wash the smear with running tap water.
  3. Pour Gram's iodine on smear and after 1 minute, wash it with tap water.
  4. Pour alcohol on the smear until the purple color no longer comes from the smear.
  5. Pour safranin on the smear and after 45 seconds, wash it with tap water and keep the smear dry in air.
  6. Observe the stained smear under oil immersion lens and note down the arrangement, shape, and Gram-reaction of the cells.

OBSERVATION

The Gram-positive bacteria appear in purple color and Gram-negative bacteria appear in pink color.

Have you ever listened about the “Second Brain”?

Yes, you have! whenever you are told to trust your gut instinct. This brain and gut connection is not just metaphorical. An extraordinarily extensive network of neurons (more than 100 million neurons) lines our gut that scientists have named it the Second Brain.

What about the inhabitants of gut including good and bad microbial flora?

Gut microbiota weighs up to 2kg containing trillions of micro-organisms. One-third of these microbiotas is common to all people while others are specific to every individual’s gut depending on the type of diet they take in and their lifestyle.

“Gut flora is a complex community of organisms that inhabit human and animal digestive system”. Relation between humans and gut flora is mutualistic. Bacteria in the digestive system assist in nutrient metabolism, vitamin production, and waste processing. They also aid in the host's immune system response to pathogenic bacteria.

Healthy Microbiota & Healthy Brain

The gut has a bidirectional relationship with the central nervous system referred to as the “gut-brain axis”. Introduction of good bacterial strain reduces anxiety and stress level. Gut-brain axis is used by bacteria to affect the brain function. The most significant factor related to the health of microbiome -- thus, brain – is healthy food. Following are the positive influencing microbiota Lactobacillus that produce lactic acid are found in yogurt. Taking in yogurt will boost mental capacity and relieve stress, it also aids in digestion and relieves constipation. But make sure yogurt is live culture (probiotic). Bifidobacteria feast on chocolate and ferment it causing positive effects on our health and body. Dark chocolate is also very beneficial for the heart because bacteria (Bifidobacterium, LAB, yeast) ferment it into healthful anti-oxidants. Prebiotic foods including raw garlic, raw, and cooked onions allow the healthy microbiota to grow and thrive while inhibits the growth of non-healthy microbiota. Environmental toxins can disturb microbiome and have adverse effects on brain health to save ourselves from these effects, use of home filtered water should be made compulsory. Such filters should be used that remove harmful toxins like chlorine. Fermented foods like pickles, kefir, kimchi etc. are the source of Lactobacillus lactis species and defend against leaky gut. These were some healthy microbial flora and their sources having a positive effect on your body and brain.

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Non-Healthy Microbiota & Whacky Gut

There are bidirectional links between stress and microbiota. Irritable Bowel Syndrome (IBS) and Chronic Fatigue Syndrome (CSF) are also related to the gut microbiota. In CSF patients there is an alteration in normal microbiota resulting in symptoms as depression, neurocognitive impairment, pain and sleep disturbance. While IBS is considered as a gut-brain disorder which is worsened by stress. Researchers are investigating whether these unhealthy microbiota resulting in IBS are also the cause of mood disorders. No bacteria can be inherited as bad, when our body is out of balance it takes advantage and proliferates. Some bacteria having a bad reputation are given below Microbial imbalance as a high level of Lactobacillus can also cause mood disturbance and sleep disturbances. Staphylococcus can cause food poisoning, it can be found in unpasteurized milk and can affect when hygiene is poor. Higher levels of the bad clostridium bacteria can cause fatigue by using bidirectional gut-brain axis. By eating junk food firmicutes and bifidobacteria level falls and there is a rise in the level of bacteroidetes causing the lethargic behavior to upshot and immunity problems set in.

Healthy Gut of a Baby

It is believed that when babies are born their guts are sterile, as soon as they encounter the genitourinary tract and mother’s skin, they are exposed to microbial flora. Microbial flora is important to develop in infants or babies for normal functioning. This healthy microbial flora to a baby is also provided by mother through breastfeeding. Milk is a cocktail of healthy microbiota and immunoglobulins causing development and growth of microbial flora in infant’s gut. So it is necessary for mothers to take healthy balanced diets rich in probiotic, prebiotic and fermented foods.

CHOLERA is a specific infectious disease that affects the lower portion of the intestine and is characterized by violent purging, vomiting, muscular cramp, suppression of urine and rapid collapse. It can a terrifying disease with massive diarrhea. The patient’s fluid losses are enormous every day with severe rapid dehydration, death comes within hours.

ETIOLOGY

  • Site: GIT (Gastrointestinal Track)
  • Agent: VIBRIO CHOLERA

MORPHOLOGICAL CHARACTER

Gram-negative, curved rods, non-capsulated, non-spore, motile by means of flagella (polar) they occur singly.

CULTURAL CHARACTER

  1. They produce smooth, convex, round, colonies which appear opaque and granular in transmitted light.
  2. They can grow on many kinds of media including enriching media contains bile salt and asparagine.
  3. They particularly grow on TCB agar (Thiosulfate Citrate Bile Salt agar) and produce yellow colonies.
  4. They are readily killed by acid and optimum pH for growth is 8.5-9.5.

BIOCHEMICAL CHARACTER

They ferment sucrose and maltose but not arabinose. They are oxidase positive which make them different from enteric Gram-negative rods. Some are halotolerant while others are halophilic require presence of NaCl for their growth.

ANTIGENIC CHARACTER

  • 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.

CHOLERA TOXIN

V. Cholera elaborated an enterotoxin that is responsible for the loss of fluid is Cholera, called CHOLERAGEN. It is a polymeric protein with a molecular weight 84,000 daltons containing two major domains. The domain "A" with molecular weight 28,000 daltons, play the key role in the biological activity of the Choleragen. The domain "B" is also known as CHOLERAGENOID with a molecular weight 56,000 daltons bind the toxin to its receptors on host cell surface. it is also the immunologically active region of the toxin.

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Vibrio Cholera has been shown to produce a second toxin called ZONULA OCCULUDENS TOXIN (ZOT). This toxin disintegrates the tight junction between enterocytes, allowing escape of water and electrolytes.

MODE OF ACTION OF TOXIN

The toxin subunit "A" and "B" promote the entry of subunit "A" into the cell, "B" subunit is responsible for attachment of toxin to the epithelial cell of the small intestine. This subunit alters the activity of the regulatory protein, that controls the activity of enzyme, Adenylate Cyclase. This enzyme converts the ATP (Adenosine Tri-Phosphate) into CAMP (Cyclic Adenosine 5 Mono Phosphate). This increase in cyclic AMP level causes loss of water, electrolytes and result in diarrhea. This may lead to death because of dehydration and acidosis.

PATHOGENESIS

Cholera occurs in epidemic form under the condition of overcrowding, floods, wars, and famine. Humans are the only known natural hosts. A person may have to ingest 108 – 1010 organism to become infected. Vibrio Cholera is transferred from one person to another by ingestion of contaminated water or foodstuff. The contact with the carrier can also contribute to epidemics.

The Cholera bacilli find their way into the small intestine where they proliferate and elaborate the Choleragen. The toxin elevates the produces a massive secretion of isotonic fluid into the lumen of the intestine.

CLINICAL FINDING

The incubation period is few hours to 4 days. After incubation, there is sudden onset of nausea, vomiting, diarrhea with abdominal cramps, rapid dehydration and loss of fluid electrolytes. Mortality rate without treatment is 25% to 50%.

LABORATORY DIAGNOSIS

Diagnosis of Cholera patient by physical examination of stool, direct microscopic examination. Culture technique and also by agglutination method.

SMEAR

Smear made from stool sample is not distinctive however darkfield microscopy or phase contrast microscopy can show motile Vibrios.

CULTURE

There is rapid growth on peptone agar, TCB’s near pH 9 colony can be picked after 18-24 hours of incubation.

AGGLUTINATION

Agglutination test using anti O group on serum and also by the biochemical reaction.

EPIDEMIOLOGY

Man is the only host of Cholera disease and spread of infection is from person to person with contaminated water, food or flies. In many intensive 1% to 5% of exposed susceptible person developed the disease. The carrier state seldom exceeds 3 to 4 weeks.

PREVENTION

  1. Good water supply. Proper treatment of water should be there before supply to the town.
  2. Proper treatment of sewerage system.
  3. Personal hygiene and proper sanitation.

TREATMENT

Individual infected with Cholera require rehydration adequately by giving a solution of Oral Rehydration Salts (ORS) containing sodium chloride, sodium bicarbonate, potassium chloride and glucose. During the epidemic, 80-90% of diarrhea patient can be treated by oral rehydration alone but the patient who becomes severely dehydrate must be given intravenous fluid.

Microscopic examination of urine is also called as the “liquid biopsy of the urinary tract”.

Urine consists of various microscopic, insoluble, solid elements in suspension. These elements are classified as organized or unorganized. Organized substances include red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites. The unorganized substances are crystalline and amorphous material. These elements are suspended in urine and on standing they settle down and sediment at the bottom of the container; therefore they are known as urinary deposits or urinary sediments. Examination of urinary deposit is helpful in diagnosis of urinary tract diseases as shown in Table 825.1.

Table 825.1 Urinary findings in renal diseases
Condition Albumin RBCs/HPF WBCs/HPF Casts/LPF Others
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.

Different types of urinary sediments are shown in Figure 825.1. The major aim of microscopic examination of urine is to identify different types of cellular elements and casts. Most crystals have little clinical significance.

Figure 825.1 Different types of urinary sediment
Figure 825.1 Different types of urinary sediment

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Specimen

The cellular elements are best preserved in acid, hypertonic urine; they deteriorate rapidly in alkaline, hypotonic solution. A mid-stream, freshly voided, first morning specimen is preferred since it is the most concentrated. The specimen should be examined within 2 hours of voiding because cells and casts degenerate upon standing at room temperature. If preservative is required, then 1 crystal of thymol or 1 drop of formalin (40%) is added to about 10 ml of urine.

Method

A well-mixed sample of urine (12 ml) is centrifuged in a centrifuge tube for 5 minutes at 1500 rpm and supernatant is poured off. The tube is tapped at the bottom to resuspend the sediment (in 0.5 ml of urine). A drop of this is placed on a glass slide and covered with a cover slip (Figure 825.2). The slide is examined immediately under the microscope using first the low power and then the high power objective. The condenser should be lowered to better visualize the elements by reducing the illumination.

Figure 825.2 Preparation of urine sediment for microscopic examination
Figure 825.2 Preparation of urine sediment for microscopic examination

CELLS

Cellular elements in urine are shown in Figure 825.3.

Figure 825.3 Cells in urine
Figure 825.3 Cells in urine (1) Isomorphic red blood cells, (2) Crenated red cells, (3) Swollen red cells, (4) Dysmorphic red cells, (5) White blood cells (pus cells), (6) Squamous epithelial cell, (7) Transitional epithelial cells, (8) Renal tubular epithelial cells, (9) Oval fat bodies, (10) Maltese cross pattern of oval fat bodies, and (11) spermatozoa

Red Blood Cells

Normally there are no or an occasional red blood cell in urine. In a fresh urine sample, red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter (called as isomorphic red cells). However, red cells may appear swollen (thin discs of greater diameter, 9-10 μ) in dilute or hypotonic urine, or may appear crenated (smaller diameter with spikey surface) in hypertonic urine. In glomerulonephritis, red cells are typically described as being dysmorphic (i.e. markedly variable in size and shape). They result from passage of red cells through the damaged glomeruli. Presence of > 80% of dysmorphic red cells is strongly suggestive of glomerular pathology.

The quantity of red cells can be reported as number of red cells per high power field.

Causes of hematuria have been listed earlier.

White Blood Cells (Pus Cells)

White blood cells are spherical, 10-15 μ in size, granular in appearance in which nuclei may be visible. Degenerated white cells are distorted, smaller, and have fewer granules. Clumps of numerous white cells are seen in infections. Presence of many white cells in urine is called as pyuria. In hypotonic urine white cells are swollen and the granules are highly refractile and show Brownian movement; such cells are called as glitter cells; large numbers are indicative of injury to urinary tract.

Normally 0-2 white cells may be seen per high power field. Pus cells greater than 10/HPF or presence of clumps is suggestive of urinary tract infection.

Increased numbers of white cells occur in fever, pyelonephritis, lower urinary tract infection, tubulointerstitial nephritis, and renal transplant rejection.

In urinary tract infection, following are usually seen in combination:

  • Clumps of pus cells or pus cells >10/HPF
  • Bacteria
  • Albuminuria
  • Positive nitrite test

Simultaneous presence of white cells and white cell casts indicates presence of renal infection (pyelonephritis).

Eosinophils (>1% of urinary leucocytes) are a characteristic feature of acute interstitial nephritis due to drug reaction (better appreciated with a Wright’s stain).

Renal Tubular Epithelial Cells

Presence of renal tubular epithelial cells is a significant finding. Increased numbers are found in conditions causing tubular damage like acute tubular necrosis, pyelonephritis, viral infection of kidney, allograft rejection, and salicylate or heavy metal poisoning.

These cells are small (about the same size or slightly larger than white blood cell), polyhedral, columnar, or oval, and have granular cytoplasm. A single, large, refractile, eccentric nucleus is often seen.

Renal tubular epithelial cells are difficult to distinguish from pus cells in unstained preparations.

Squamous Epithelial Cells

Squamous epithelial cells line the lower urethra and vagina. They are best seen under low power objective (×10). Presence of large numbers of squamous cells in urine indicates contamination of urine with vaginal fluid. These are large cells, rectangular in shape, flat with abundant cytoplasm and a small, central nucleus.

Transitional Epithelial Cells

Transitional cells line renal pelvis, ureters, urinary bladder, and upper urethra. These cells are large, and diamond- or pear-shaped (caudate cells). Large numbers or sheets of these cells in urine occur after catheterization and in transitional cell carcinoma.

Oval Fat Bodies

These are degenerated renal tubular epithelial cells filled with highly refractile lipid (cholesterol) droplets. Under polarized light, they show a characteristic “Maltese cross” pattern. They can be stained with a fat stain such as Sudan III or Oil Red O. They are seen in nephrotic syndrome in which there is lipiduria.

Spermatozoa

They may sometimes be seen in urine of men.

Telescoped urinary sediment: This refers to urinary sediment consisting of red blood cells, white blood cells, oval fat bodies, and all types of casts in roughly equal proportion. It occurs in lupus nephritis, malignant hypertension, rapidly proliferative glomerulonephritis, and diabetic glomerulosclerosis.

ORGANISMS

Organisms detectable in urine are shown in Figure 825.4.

Figure 825.4 Organisms in urine
Figure 825.4 Organisms in urine: (A) Bacteria, (B) Yeasts, (C) Trichomonas, and (D) Egg of Schistosoma haematobium

Bacteria

Bacteria in urine can be detected by microscopic examination, reagent strip tests for significant bacteriuria (nitrite test, leucocyte esterase test), and culture.

Significant bacteriuria exists when there are >105 bacterial colony forming units/ml of urine in a cleancatch midstream sample, >104 colony forming units/ml of urine in catheterized sample, and >103 colonyforming units/ml of urine in a suprapubic aspiration sample.

  1. 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.
  2. Chemical or reagent strip tests for significant bacteriuria: These are given earlier.
  3. 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.

If three or more species of bacteria are identified on culture, it almost always indicates contamination by vaginal flora.

Negative culture in the presence of pyuria (‘sterile’ pyuria) occurs with prior antibiotic therapy, renal tuberculosis, prostatitis, renal calculi, catheterization, fever in children (irrespective of cause), female genital tract infection, and non-specific urethritis in males.

Yeast Cells (Candida)

These are round or oval structures of approximately the same size as red blood cells. In contrast to red cells, they show budding, are oval and more refractile, and are not soluble in 2% acetic acid.

Presence of Candida in urine may suggest immunocompromised state, vaginal candidiasis, or diabetes mellitus. Usually pyuria is present if there is infection by Candida. Candida may also be a contaminant in the sample and therefore urine sample must be examined in a fresh state.

Trichomonas vaginalis

These are motile organisms with pear shape, undulating membrane on one side, and four flagellae. They cause vaginitis in females and are thus contaminants in urine. They are easily detected in fresh urine due to their motility.

Eggs of Schistosoma haematobium

Infection by this organism is prevalent in Egypt.

Microfilariae

They may be seen in urine in chyluria due to rupture of a urogenital lymphatic vessel.

CASTS

Urinary casts are cylindrical, cigar-shaped microscopic structures that form in distal renal tubules and collecting ducts. They take the shape and diameter of the lumina (molds or ‘casts’) of the renal tubules. They have parallel sides and rounded ends. Their length and width may be variable. Casts are basically composed of a precipitate of a protein that is secreted by tubules (Tamm-Horsfall protein). Since casts form only in renal tubules their presence is indicative of disease of the renal parenchyma. Although there are several types of casts, all urine casts are basically hyaline; various types of casts are formed when different elements get deposited on the hyaline material (Figure 825.5). Casts are best seen under low power objective (×10) with condenser lowered down to reduce the illumination.

Figure 825.5 Genesis of casts in urine
 Figure 825.5 Genesis of casts in urine. All cellular casts degenerate to granular and waxy casts

Casts are the only elements in the urinary sediment that are specifically of renal origin.

Casts (Figure 825.6) are of two main types:

  1. Noncellular: Hyaline, granular, waxy, fatty
  2. Cellular: Red blood cell, white blood cell, renal tubular epithelial cell.

Hyaline and granular casts may appear in normal or diseased states. All other casts are found in kidney diseases.

Figure 825.6 Urinary casts
Figure 825.6 Urinary casts: (A) Hyaline cast, (B) Granular cast, (C) Waxy cast, (D) Fatty cast, (E) Red cell cast, (F) White cell cast, and (G) Epithelial cast

Non-cellular Casts

Hyaline casts: These are the most common type of casts in urine and are homogenous, colorless, transparent, and refractile. They are cylindrical with parallel sides and blunt, rounded ends and low refractive index. Presence of occasional hyaline cast is considered as normal. Their presence in increased numbers (“cylinduria”) is abnormal. They are composed primarily of Tamm-Horsfall protein. They occur transiently after strenuous muscle exercise in healthy persons and during fever. Increased numbers are found in conditions causing glomerular proteinuria.

Granular casts: Presence of degenerated cellular debris in a cast makes it granular in appearance. These are cylindrical structures with coarse or fine granules (which represent degenerated renal tubular epithelial cells) embedded in Tamm-Horsfall protein matrix. They are seen after strenuous muscle exercise and in fever, acute glomerulonephritis, and pyelonephritis.

Waxy cast: These are the most easily recognized of all casts. They form when hyaline casts remain in renal tubules for long time (prolonged stasis). They have homogenous, smooth glassy appearance, cracked or serrated margins and irregular broken-off ends. The ends are straight and sharp and not rounded as in other casts. They are light yellow in color. They are most commonly seen in end-stage renal failure.

Fatty casts: These are cylindrical structures filled with highly refractile fat globules (triglycerides and cholesterol esters) in Tamm-Horsfall protein matrix. They are seen in nephrotic syndrome.

Broad casts: Broad casts form in dilated distal tubules and are seen in chronic renal failure and severe renal tubular obstruction. Both waxy and broad casts are associated with poor prognosis.

Cellular Casts

To be called as cellular, casts should contain at least three cells in the matrix. Cellular casts are named according to the type of cells entrapped in the matrix.

Red cell casts: These are cylindrical structures with red cells in Tamm-Horsfall protein matrix. They may appear brown in color due to hemoglobin pigmentation. These have greater diagnostic importance than any other cast. If present, they help to differentiate hematuria due to glomerular disease from hematuria due to other causes. RBC casts usually denote glomerular pathology e.g. acute glomerulonephritis.

White cell casts: These are cylindrical structures with white blood cells embedded in Tamm-Horsfall protein matrix. Leucocytes usually enter into tubules from the interstitium and therefore presence of leucocyte casts indicates tubulointerstitial disease like pyelonephritis.

Renal tubular epithelial cell casts: These are composed of renal tubular epithelial cells that have been sloughed off. They are seen in acute tubular necrosis, viral renal disease, heavy metal poisoning, and acute allograft rejection. Even an occasional renal tubular cast is a significant finding.

CRYSTALS

Crystals are refractile structures with a definite geometric shape due to orderly 3-dimensional arrangement of its atoms and molecules. Amorphous material (or deposit) has no definite shape and is commonly seen in the form of granular aggregates or clumps.

Crystals in urine (Figure 825.7) can be divided into two main types: (1) Normal (seen in normal urinary sediment), and (2) Abnormal (seen in diseased states).

Figure 825.7 Crystals in urine
Figure 825.7 Crystals in urine. (A) Normal crystals: (1) Calcium oxalate, (2) Triple phosphates, (3) Uric acid, (4) Amorphous phosphates, (5) Amorphous urates, (6) Ammonium urate. (B) Abnormal crystals: (1) Cysteine, (2) Cholesterol, (3) Bilirubin, (4) Tyrosine, (5) Sulfonamide, and (6) Leucine

However, crystals found in normal urine can also be seen in some diseases in increased numbers.

Most crystals have no clinical importance (particularly phosphates, urates, and oxalates). Crystals can be identified in urine by their morphology. However, before reporting presence of any abnormal crystals, it is necessary to confirm them by chemical tests.

Normal Crystals

Crystals present in acid urine:

  1. 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.
  2. 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.
  3. 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.

Crystals present in alkaline urine:

  1. 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.
  2. 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.
  3. 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.

Abnormal Crystals

They are rare, but result from a pathological process.

These occur in acid pH, often in large amounts. Abnormal crystals should not be reported on microscopy alone; additional chemical tests are done for confirmation.

  1. 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.
  2. 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.
  3. 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.
  4. 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).
  5. 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.
  6. Sulfonamide crystals: They are variably shaped crystals, but usually appear as sheaves of needles. They occur following sulfonamide therapy. They are soluble in acetone.
Routine care and proper maintenance of microscope will ensure good performance over the years. In addition to this, a properly maintained and clean microscope will always be ready for use at any time. Professional cleaning and maintenance should be considered when routine techniques fail to produce optimal performance of the microscope.
 
Cleaning and maintenance supplies
 
Dust cover: When not in use, a microscope should be covered to protect it from dust, hair, and any other possible sources of dirt. It is important to note that a dust cover should never be placed over a microscope while the illuminator is still on.
 
Lens tissue: Lint-free lens tissues are delicate wipes that would not scratch the surface of the oculars or objective. Always ensure that you are using these types of tissues. Never substitute facial tissue or paper towel, as they are too abrasive.
 
Lens cleaner: Lens cleaning solution assists in removing fingerprints and smudges on lenses and objectives. Apply the lens cleaner to the lens tissue paper and clean/polish the surface.
 
Compressed air duster: Using compressed air to rid the microscope of dust particles is far superior to using your own breath and blowing onto the microscope. Compressed air is clean, and avoids possible contamination of saliva particles.
 
Maintenance tips
 
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
A blood smear is examined for:
 
 
A peripheral blood smear has three parts: Head, body, and tail. Also read: PREPARATION OF BLOOD SMEAR BY WEDGE METHOD.
 
A blood smear should be examined in an orderly manner. Initially, blood smear should be observed under low power objective (10×) to assess whether the film is properly spread and stained, to assess cell distribution, and to select an area for examination of blood cells. Best morphologic details are seen in the area where red cells are just touching one another. Low power view is also helpful for the identification of Rouleaux formation, autoagglutination of red cells, and microfilaria. High power objective (45×) is suitable for examination of red cell morphology and for differential leukocyte count. A rough estimate of total leukocyte count can be obtained which also serves to crosscheck the total leukocyte count done by manual counting or automated method. Oil-immersion objective (100×) is used for more detailed examination of any abnormal cells.
 
Further Reading:
 
Box 802.1 Role of blood smear in thrombocytopeniaPlatelets are small, 1-3 μm in diameter, purple structures with tiny irregular projections on surface. In blood films prepared from non-anticoagulated blood (i.e. direct fingerstick), they occur in clumps. If platelet count is done on automated blood cell counters using EDTA-anticoagulated blood sample, about 1% of persons show falsely low count due to the presence in them of EDTA dependent antiplatelet antibody. Examination of a parallel blood film is useful in avoiding the false diagnosis of thrombocytopenia in such cases. Occasionally, platelets show rosetting around neutrophils (platelet satellitism) (see Figure 802.1). This is seen in patients with platelet antibodies and in apparently normal persons. Blood smear examination can be helpful in determining underlying cause of thrombocytopenia such as leukemia, lymphoma, or microangiopathic hemolytic anemia (Box 802.1).
 
Also Read:
 
For meaningful interpretation, absolute count of leukocytes should be reported. These are obtained as follows:
 
Absolute Leukocyte Count = Leukocyte% × Total Leukocyte Count/ml
 
 
Neutrophilia:
 
An absolute neutrophil count greater than 7500/μl is termed as neutrophilia or neutrophilic leukocytosis.
 
Causes
 
  1. Acute bacterial infections: Abscess, pneumonia, meningitis, septicemia, acute rheumatic fever, urinary tract infection.
  2. Tissue necrosis: Burns, injury, myocardial infarction.
  3. Acute blood loss
  4. Acute hemorrhage
  5. Myeloproliferative disorders
  6. Metabolic disorders: Uremia, acidosis, gout
  7. Poisoning
  8. Malignant tumors
  9. Physiologic causes: Exercise, labor, pregnancy, emotional stress.
 
Leukemoid reaction: This refers to the presence of markedly increased total leukocyte count (>50,000/cmm) with immature cells in peripheral blood resembling leukaemia but occurring in non-leukemic disorders (see Figure 801.2). Its causes are:
 
  • Severe bacterial infections, e.g. septicemia, pneumonia
  • Severe hemorrhage
  • Severe acute hemolysis
  • Poisoning
  • Burns
  • Carcinoma metastatic to bone marrow Leukemoid reaction should be differentiated from chronic myeloid leukemia (Table 801.1).
 
Table 801.1 Differences between leukemoid reaction and leukemia
Table 801.1 Differences between leukemoid reaction and leukemia
 
Figure 801.2 Leukemoid reaction in blood smear
Figure 801.2 Leukemoid reaction in blood smear
 
 
Absolute neutrophil count less than 2000/μl is neutropenia. It is graded as mild (2000-1000/μl), moderate (1000-500/μl), and severe (< 500/μl).
 
Causes
 
I. Decreased or ineffective production in bone marrow:
 
  1. Infections 
    (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
  2. Hematologic disorders: megaloblastic anemia, aplastic anemia, aleukemic leukemia, myelophthisis.
  3. Drugs:
    (a) Idiosyncratic action: Analgesics, antibiotics, sulfonamides, phenothiazines, antithyroid drugs, anticonvulsants.
    (b) Dose-related: Anticancer drugs
  4. Ionizing radiation
  5. Congenital disorders: Kostman's syndrome, cyclic neutropenia, reticular dysgenesis.
 
II. Increased destruction in peripheral blood:
 
  1. Neonatal isoimmune neutropaenia
  2. Systemic lupus erythematosus
  3. Felty's syndrome
 
III. Increased sequestration in spleen:
 
  1. Hypersplenism
 
Eosinophilia:
 
This refers to absolute eosinophil count greater than 600/μl.
 
Causes
 
  1. Allergic diseases: Bronchial asthma, rhinitis, urticaria, drugs.
  2. Skin diseases: Eczema, pemphigus, dermatitis herpetiformis.
  3. Parasitic infection with tissue invasion: Filariasis, trichinosis, echinococcosis.
  4. Hematologic disorders: Chronic Myeloproliferative disorders, Hodgkin's disease, peripheral T cell lymphoma.
  5. Carcinoma with necrosis.
  6. Radiation therapy.
  7. Lung diseases: Loeffler's syndrome, tropical eosinophilia
  8. Hypereosinophilic syndrome.
 
Basophilia:
 
Increased numbers of basophils in blood (>100/μl) occurs in chronic myeloid leukemia, polycythemia vera, idiopathic myelofibrosis, basophilic leukemia, myxedema, and hypersensitivity to food or drugs.
 
Monocytosis:
 
This is an increase in the absolute monocyte count above 1000/μl.
 
Causes
 
  1. Infections: Tuberculosis, subacute bacterial endocarditis, malaria, kala azar.
  2. Recovery from neutropenia.
  3. Autoimmune disorders.
  4. Hematologic diseases: Myeloproliferative disorders, monocytic leukemia, Hodgkin's disease.
  5. Others: Chronic ulcerative colitis, Crohn's disease, sarcoidosis.
 
Lymphocytosis:
 
Box 801.1 Differential diagnosis of LymphocytosisThis is an increase in absolute lymphocyte count above upper limit of normal for age (4000/μl in adults, >7200/μl in adolescents, >9000/μl in children and infants) (Box 801.1).
 
Causes
 
  1. Infections: 
    (a) Viral: Acute infectious lymphocytosis, infective hepatitis, cytomegalovirus, mumps, rubella, varicella
    (b) Bacterial: Pertussis, tuberculosis
    (c) Protozoal: Toxoplasmosis
  2. Hematological disorders: Acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, lymphoma.
  3. Other: Serum sickness, post-vaccination, drug reactions.
Approximate idea about total leukocyte count can be gained from the examination of the smear under high power objective (40× or 45×). A differential leukocyte count should be carried out. Abnormal appearing white cells are evaluated under oil-immersion objective.
 
Morphology of normal leukocytes (see Figure 800.1):
 
  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
 
Figure 800.1 Normal mature white blood cells in peripheral blood
Figure 800.1 Normal mature white blood cells in peripheral blood
 
Morphology of abnormal leukocytes:
 
  1. Box 800.1 Role of blood smear in leukemiaToxic granules: These are darkly staining, bluepurple, coarse granules in the cytoplasm of neutrophils. They are commonly seen in severe bacterial infections.
  2. 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.
  3. Cytoplasmic vacuoles: Vacuoles in neutrophils are indicative of phagocytosis and are seen in bacterial infections.
  4. 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.
  5. 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.
  6. 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).
  7. 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.
  8. 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).
 
Figure 800.2 Morphological abnormalities of white blood cells
Figure 800.2 Morphological abnormalities of white blood cells: (A) Toxic granules; (B) Döhle inclusion body; (C) Shift to left in neutrophil series; (D) Hypersegmented neutrophil in megaloblastic anemia; (E) Atypical lymphocyte in infectious mononucleosis; (F) Blast cell in acute leukemia
 
Further Reading:
 

The microscope is the most important piece of equipment in the clinic laboratory. The microscope is used to review fecal, urine, blood, and cytology samples on a daily basis (see Figure). Understanding how the microscope functions, how it operates, and how to care for it will improve the reliability of your results and prolong the life of this valuable piece of equipment.

Parts and functions of a compound microscope

(A) Arm

Used to carry the microscope.

(B) Base

Supports the microscope and houses the light source.

(C) Oculars (or eyepieces)

The lens of the microscope you look through. The ocular also magnifies the image. The total magnification can be calculated by multiplying the objective power by the ocular power. Oculars come in different magnifications, but 10× magnification is common.

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(D) Diopter adjustment

The purpose of the diopter adjustment is to correct the differences in vision an individual may have between their left and right eyes.

(E) Interpupillary adjustment

This allows the oculars to move closer or further away from one another to match the width of an individual’s eyes. When looking through the microscope, one should see only a single field of view. When viewing a sample, always use both eyes. Using one eye can cause eye strain over a period of time.

(F) Nosepiece

The nosepiece holds the objective lenses. The objectives are mounted on a rotating turret so they can be moved into place as needed. Most nosepieces can hold up to five objectives.

(G) Objective lenses

The objective lens is the lens closest to the object being viewed, and its function is to magnify it. Objective lenses are available in many powers, but 4×, 10×, 40×, and 100× are standard. 4× objective is used mainly for scanning. 10× objective is considered “low power,” 40× is “high power” and 100× objective is referred to as “oil immersion.” Once magnified by the objective lens, the image is viewed through the oculars, which magnify it further. Total magnification can be calculated by multiplying the objective power by the ocular lens power.

For example: 100× objective lens with 10× oculars = 1000× total magnification.

Compound Microscope

(H) Stage

The platform on which the slide or object is placed for viewing.

(I) Stage brackets

Spring-loaded brackets, or clips, hold the slide or specimen in place on the stage.

(J) Stage control knobs

Located just below the stage are the stage control knobs. These knobs move the slide or specimen either horizontally (x-axis) or vertically (y-axis) when it is being viewed.

(K) Condenser

The condenser is located under the stage. As light travels from the illuminator, it passes through the condenser, where it is focused and directed at the specimen.

(L) Condenser control knob

Allows the condenser to be raised or lowered.

(M) Condenser centering screws:

These crews center the condenser, and therefore the beam of light. Generally, they do not need much adjustment unless the microscope is moved or transported frequently.

(N) Iris diaphragm

This structure controls the amount of light that reaches the specimen. Opening and closing the iris diaphragm adjusts the diameter of the light beam.

(O) Coarse and fine focus adjustment knobs

These knobs bring the object into focus by raising and lowering the stage. Care should be taken when adjusting the stage height. When a higher power objective is in place (100× objective for example), there is a risk of raising the stage and slide and hitting the objective lens. This can break the slide and scratch the lens surface. Coarse adjustment is used for finding focus under low power and adjusting the stage height. Fine adjustment is used for more delicate, high power adjustment that would require fine tuning.

(P) Illuminator

The illuminator is the light source for the microscope, usually situated in the base. The brightness of the light from the illuminator can be adjusted to suit your preference and the object you are viewing.

What is Kohler illumination?

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

Materials required

  • Specimen slide (will need tofocus under 10× power)
  • Compound microscope.

Kohler illumination

  1. Mount the specimen slide onthe stage and focus under 10×.
  2. Close the iris diaphragm completely.
  3. If the ball of light is not in the center, use the condenser centering screws to move it so that it is centered.
  4. 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).
  5. Open the iris diaphragm until the entire field is illuminated.
Note the blurry edges of the unfocused light
Figure 797.1 Note the blurry edges of the unfocused light

Adjusting the condenser height sharpens the edges of the ball of light
Figure 797.2 Adjusting the condenser height sharpens the edges of the “ball of light.”

When should you set/check Kohler?

  • During regular microscope maintenance
  • After the microscope is moved/transported
  • Whenever you suspect objects do not appear as sharp as they could be.

Further Reading:

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".

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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.

Description: Wallach’s Interpretation of Diagnostic Tests, now in its Ninth Edition, has been completely revised and updated by a new author team from the Department of Hospital Laboratories, UMass Memorial Medical Center faculty, who are carrying on the tradition of Jacques Wallach’s teachings. This text serves as a practical guide to the use of laboratory tests which aids physicians in using tests more effectively and efficiently by offering test outcomes, possible meanings, differential diagnosis, and summaries of tests available.

The book has been reorganized into 2 sections. The first section is devoted to an alphabetical listing of laboratory tests while stressing the integration of the clinical laboratory in the clinical decision making process. Test sensitivity, specificity, and positive and negative infectious disease probabilities are included whenever appropriate. Microbiology tests are listed in a separate chapter. The second section is devoted to disease states. Where appropriate, a patient’s chief complaint and/or physical findings are initially presented with subsequent discussions focused on discrete disease states as they relate to a patient’s chief complaint. Current molecular diagnostic testing, cytogenetics, common pitfalls, test limitations, and identification of appropriate tests for specific clinical presentations are also addressed.

Ninth Edition highlights include:
Detailed listing and description of routine and esoteric tests listed alphabetically, with information on when to order and how to interpret the test results based on evidence-based laboratory medicine.
Information on how to work up patients with specific symptoms and the appropriate lab tests to order
Up-to-date test procedures including molecular diagnostic tests
Detailed microbiology chapter of infectious diseases

Objective: To test organism's ability to tolerate various osmotic concentrations.

Test Procedure
1. Use a sterile loop or needle to inoculate broth tubes with different salt concentrations.
2. Incubate at the optimum temperature for 48-96 hours.

• Interpretation
Positive = growth; Negative = no growth

Objective: To test the organism's susceptibility to antibiotic penicillin.

Test Procedure and Interpretation: See the Optochin Disc Test.

Discovery of Penicillin

The discovery of penicillin's antibiotic powers is attributed to Alexander Fleming. The story goes that he returned to his laboratory one day in September 1928 to find a Petri dish containing Staphylococcus bacteria with its lid removed.

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The dish had become contaminated by blue-green mold. He noted that there was a clear ring surrounding the mold where the bacteria had been inhibited from growing.

This discovery of the mold - Penicillium notatum - and his recognition of its special powers set the wheels in motion to create one of the most used drugs in medical history.

In March 1942, Anne Miller became the first civilian to be treated successfully with penicillin having almost died from a huge infection following a miscarriage.

Although Fleming often gets the accolade for having invented the first antibiotic, there was a lot of work to do before penicillin could become as commonly used and useful as it is today.

The bulk of the work was eventually carried out by scientists who had a much better-stocked laboratory and a deeper understanding of chemistry than Fleming. Dr. Howard Florey, Dr. Norman Heatley, and Dr. Ernst Chain carried out the first in-depth and focused studies.

Interestingly, and with impressive foresight, Fleming's Nobel Prize acceptance speech warned that the overuse of penicillin might, one day, lead to bacterial resistance.

Objective: To determine the organism's oxygen requirement.

Test Procedure
1. Inoculate 5 ml of BHI broth with your unknown organism and incubate overnight. We have found that broth cultures provide much more accurate results than using inoculum from a plate. However, if you are inoculating from a plate, make sure you use a very light inoculum.
2. Obtain a thioglycollate tube and make sure that it does not have more than 20% of the medium in pink color. This may happen due to oxidation of the top layer of the medium. To restore anaerobic conditions, such a tube should be placed in boiling water for 10 minutes and then cooled to room temperature. If you do not see any pink color against a white background, the tube is good to use.
3. Use a sterile narrow thin needle (rather than a thick one), insert into your culture broth and slowly stab a thioglycollate tube to the bottom. Carefully remove the needle along the same stab line. Do not shake the tube or move the needle around, or you will introduce extra oxygen into the medium. The needle should reach all the way to the bottom of the tube.
4. Incubate the tube at 30°C (without any regard to the optimum temperature requirement of your species) for 24 hours before reading the tube.

• Interpretation
-- Aerobe: band of growth on the top of the tube. Some species have a tendency to grow very rapidly in thioglycollate tube so that the growth covers a rather thick band from the top and extends to the line of stab where there is oxygen available (brought in by the needle). So it is best to look at the bottom 1-cm of the tube and if it is clear with no growth whatsoever, then you can be sure that you have an aerobe.
-- Microaerophile: band of no growth at the top, then a band of growth extending a short distance down proceeded by no growth to the bottom. The bottom 3-cm of the tube should be clear of any growth.
-- Facultative Anaerobe: growth can occur either throughout the tube or can begin at some point below the surface and extend all the way to the bottom, even in the 1-cm bottom of the tube.
-- Anaerobe: growth only at the bottom fifth of the tube.

OXIDASE TEST

  • 05 Apr 2017

Objective: To determine the presence of the oxidase enzymes (e.g. cytochrome c oxidase).

Test Procedure and Interpretation
1. Grow the culture on a BHI plate for 48 hours. Up to 7 day old cultures are fine.
2. Warm the plate to 20-37°C. Pick a good amount of the test organism with a sterile swab and rub onto the reaction area of a DrySlide card. If the organism is oxidase positive, a purple color will develop on the slide within 20 seconds. The slide is saturated with Kovacs' oxidase reagent (1% N, N, N', N' tetra-methyl-p-phenylene diamine dihydrochloride). Oxidase negative colonies do not change the color of the slide in 20 seconds, or if they do, it would be after 20 seconds and thus negative.

• Precautions
-- Most Gram-positive bacteria and all Enterobacteriaceae are oxidase negative.
-- Do not attempt to perform an oxidase test on any colonies growing on media containing glucose, as glucose fermentation will inhibit oxidase enzyme activity, and result in possible false negatives. Oxidase test on Gram-negative rods should be performed only on colonies from nonselective and/or non-differential media to ensure valid results.
-- The culture should not be older than a week, unless the species is a slowgrower. False results may be obtained if the culture is old.
-- The oxidase reagent quickly auto-oxidizes (by free oxygen in the air) and loses its sensitivity. The reagent should be discarded if any precipitate forms. Avoid undue exposure of the reagent to light. The reagent must be made up fresh each week.
-- Time period for color development must be adhered to since a purpleblack color may develop later due to auto-oxidation of reagent and/or a weak positive oxidase organism containing a small quantity of cytochrome c oxidase.
-- As an alternative to Kovacs' reagent, one may use a few drops of a 1:1 mixture of 1% α−naphthol in 95% ethanol and freshly prepared 1% aqueous dimethyl-p-phenylenediamine oxalate.

Objective: To test an organism's susceptibility to the chemical, optochin. Optochin susceptibility tests the fragility of the bacterial cell membrane. This test is mainly used to differentiate between Streptococcus pneumoniae (sensitive) and other Streptococcus species (resistant)

Test Procedure
1. Pick a single pure colony with a sterile swab to inoculate a SBA plate. Streak the entire blood agar plate with the swab. Turn plate 90 degrees and re-streak with the same swab. Blood agar plate must be used for optochin testing since all species of Streptococcus are fastidious organisms and require extra enrichment for growth.
2. With alcohol flamed forceps, aseptically remove an optochin disc and apply to the center of the plate. Gently apply pressure to disc so that it adheres to the surface of the plate but do not press disc down into the medium.
3. Invert plate and incubate for 48 hours at your organism's optimum growth temperature.

• Interpretation
-- Sensitive (S): A distinct zone of inhibition (5 to 30 mm) with a clear-cut margin around disc.
-- Resistant (R): Growth not inhibited around disc.

• Precautions
-- Occasionally, a few scattered optochin resistant colonies of S. pneumoniae may be observed in a wide zone of inhibition.
-- Occasionally an alpha-Streptococcus spp. may exhibit a very small zone (1 to 2 mm) of inhibition. S. pneumoniae exhibits a zone of inhibition at least 5 mm or greater in diameter.

Objective: To test an organism's susceptibility to the antibiotic novobiocin.

Test Procedure
1. Streak a BHI plate using a sterile cotton swab. Turn the plate 90 degrees and restreak with the same swab
2. Using a pair of alcohol flamed forceps, aseptically place a novobiocin disc in the center of the plate. Apply gentle pressure to disc so it adheres to the surface of the agar but try not to press too much to embed the disc into the agar.
3. Incubate the inverted plate 48 hours at your organism's optimum growth temperature.

• Interpretation
Sensitive (S): No growth around disc; clear zone around disc.
Resistant (R): Growth not inhibited; growth around disc.

MOTILITY TEST

  • 05 Apr 2017
Objective: To determine whether an organism is motile.
 
Test Procedure and Interpretation
 
1. Prepare a semisolid agar medium in a test tube.
2. Inoculate with a straight wire, making a single stab down the center of the tube to about half the depth of the medium.
3. Incubate under the conditions favoring motility.
4. Incubate at 37°C
5. Examine at intervals, e.g. after 6 h, and 1 and 2 days (depends on generation time of bacteria) . Freshly prepared medium containing 1% glucose can be used for motility tests on anaerobes.
 
Results: Hold the tube up to the light and look at the stab line to determine motility.
 
Non-motile bacteria generally give growths that are confined to the stab-line, have sharply defined margins and leave the surrounding medium clearly transparent.
Motile Bacteria typically give diffuse, hazy growths that spread throughout the medium rendering it slightly opaque.

Objective: To determine the ability of an organism to grow in 7.5% NaCl and ferment mannitol.

Test Procedure
1. Streak an MSA plate with a light line of inoculum from the pure culture of the test organism using a sterile loop.
2. Incubate at 30°C for at least 48 hours.

• Interpretation
Any significant growth indicates the organism is a Staphylococcus species. The phenol red indicator changes to yellow at low (acid) pH, which is a product of fermentation. Therefore, fermentation of mannitol will change the color of agar to yellow. Orange is negative.
Positive: Growth, yellow color (mannitol "+").
Negative: Growth or no growth; red or orange color (mannitol "-").

Objective: Some pathogens are able to produce exoenzymes called hemolysins which lyse red blood cells and thus their action can be demonstrated on a blood agar plate.

Test Procedure
1. Using a sterile loop, inoculate a blood plate (SBA) with the pure culture of the organism to be tested using the quadrant method. Also stab the medium in the second quadrant with your loop. (Some hemolysins show their effects better under lower oxygen concentrations.)
2. Incubate for 48 hours at optimum temperature for the organism.

• Interpretation
Interpret by noting the reaction around isolated colonies as follows:
Alpha (α) hemolysis: formation of a green or brown zone around the colonies (due to loss of potassium from the red cells).
Beta (β) hemolysis: complete lysis of cells and reduction of released hemoglobin; a clear zone appears around isolated colonies.
Gamma (γ) hemolysis: no hemolytic reaction (no change of the medium surrounding isolated colonies).

• Precautions
-- The reaction should be checked only around isolated colonies. If you do not have isolated colonies on the blood agar, a lighter inoculation should be streaked and the test repeated.

DNASE TEST

  • 05 Apr 2017

Objective: DNase mediates the hydrolysis of DNA. Methyl Green indicator is stable at pHs above 7.5 but becomes colorless at lower pHs. The hydrolysis of DNA in the agar by bacterial DNase reduces the agar pH.

Test Procedure
1. Using a sterile loop, inoculate a DNA+Methyl Green agar plate with the fresh bacterial culture. Use a heavy streak line for each bacterial strain to be tested. Be sure to label the plate bottom properly for each strain.
2. Incubate at 37°C for 48 hrs.

• Interpretation
-- The test is positive if clearing develops around the areas of growth. If the color of the agar around the growth is unchanged, the test is negative (i.e., the organism is not able to produce DNase).

Objective: To determine the ability of an organism to ferment (degrade) a specific carbohydrate in a basal medium producing acid or acid with visible gas. The acid would change the color of the medium in a positive test. The following carbohydrate semi-solid media tubes are available at our lab:

- Arabinose
- Glucose
- Glycerol
- Inulin
- Maltose
- Sorbitol
- Trehalose
- Xylose

Test Procedure
1. Using a sterile needle, stab the tube within 1/4 inch of the bottom with medium inoculation.
2. Incubate for at least 48 hrs. Bacteria that are known to be slow growers should be given up to 96 hours.

• Interpretation:
-- Fermentation
Positive: Any yellow color (not orange). It does not necessarily have to be the whole tube. A positive result is referred to as ("+") or (A) or (Acid), as fermentation forms acidic products.
Negative: A red, pink or orange color - no yellow at all.
-- Gas production
Positive: Significant bubbling in semisolid medium (one small bubble is generally negative, caused by the stab). Gas may also cause the medium to get separated from tube. Record as (G) for positive gas production.
Negative: No gas bubbles except those produced by stabbing.

Objective: To determine if the organism is capable of breaking down starch into maltose through the activity of the extra-cellular α-amylase enzyme.

Test procedure
1. Use a sterile swab or a sterile loop to pick a few colonies from your pure culture plate. Streak a starch plate in the form of a line across the width of the plate. Several cultures can be tested on a single agar plate, each represented by a line or the plate may be divided into four quadrants (pie plate) for this purpose.
2. Incubate plate at 37 °C for 48 hours.
3. Add 2-3 drops of 10% iodine solution directly onto the edge of colonies. Wait 10-15 minutes and record the results.

Amylase Test

• Interpretation:
-- Positive test ("+"): The medium will turn dark. However, areas surrounding isolated colonies where starch has been hydrolyzed by amylase will appear clear.
-- Negative test ("-"): The medium will be colored dark, right up to the edge of isolated colonies.

Many different tests have been devised over the years for classification of microorganisms into families, genera, species and even subspecies. Some of these tests are quite simple to perform while others are complicated and may require sophisticated equipment. The tests presented here are among the easier ones that are utilized in major clinical laboratories around the world. These tests are ordered alphabetically in this section. Make sure that you read the complete discussion of each test before you start to perform it.

IMPORTANT: Many of the tests mentioned in the following sections are enzymatic reactions. Therefore to get a correct results, one needs to warm up the culture and all test materials to temperatures between 25-40°C for the reactions to proceed. If you have stored your plates, broth culture or test reagents in the refrigerator, you may need to place them at the 37°C incubator for 15-20 minutes before performing the test.

Objective: To determine the ability of an organism to reduce nitrate to nitrite which is then reduced to free nitrogen gas. The nitrogen in nitrate serves as an electron acceptor. The result of the denitrification process is the production of nitrite:

nitratase 1

We can test for the presence of NO2- using Reagents A and B as described below.
Denitrification may also produce N2 gas:

nitratase 2

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In this case, all the NO3- will be converted to N2 gas which escapes to the atmosphere. We can test for this step by looking for the absence of NO3- through the addition of Zn powder as described below.

Test Procedure
1. Inoculate a nitrate agar slant with your pure culture using a sterile loop to transfer a rather heavy inoculum.
2. Incubate at 37°C for at least 48 hours.
3. Add 2-3 drops of Reagent A and 2-3 drops of Reagent B to your tube. Reagent A is 0.8% sulfanilic acid in 30% acetic acid and Reagent B is 0.6% N,N-dimethyl-α-naphthylamine in 30% acetic acid (CAUTION: Reagent B is a potential carcinogen, so work in the hood and avoid inhaling it or allowing for contact with skin; wash hands thoroughly after work).

• Interpretation
Reduction of nitrate to nitrite is indicated if a red color develops quickly (within 1-2 minutes). If no color develops, add a very small amount of zinc powder (~20 mg) to the tube containing the reagents. If a pink to dark red color develops after adding the zinc powder within 5 min., the test is negative (nitrate is present and is not reduced by the organism but zinc has reduced it to nitrite). If no color develops, the test is positive (the organism was able to reduce all the nitrate to nitrite and further to N2 which escaped from the tube).

Precautions
-- If tubes are stored in the refrigerator, they should first be brought back up to the optimum temperature of the growth condition of the organism.
-- When performing the nitrate reduction test using α-naphthylamine, the color produced in a positive reaction may fade quickly. Interpret results immediately, particularly when performing a number of tests.
-- A strong nitrate-reducing organism may exhibit a brown precipitate immediately after the addition of the reagents. This is due to the effect of excess nitrite upon the p-amino group of the azo-dye and may be reduced by using dimethyl-α-naphthylamine.
-- Some organisms are capable of reducing nitrate to nitrite, yet they destroy the nitrite as fast as it is formed, yielding a false negative result. This nitrite destruction is evident in quite a few bacteria, particularly some Salmonella and Pseudomonas spp. and in Brucella suis.
-- Do not use an excess of zinc; if too much Zn is added, the large amount of hydrogen gas produced may reduce the nitrite (formed from unreduced nitrate) to ammonia (NH3) that could result in a false negative reaction or just a fleeting color reaction.

CATALASE TEST

  • 03 Apr 2017

Objective: To test for the presence of the enzyme catalase.

Catalase Test

Test Procedure
1. If the plate is refrigerated, it should be allowed to warm up to room temperature and then incubated for 15 min at 37°C before performing the test. Pick a loopful of colonies from a not-too-old pure culture plate and place on a clean glass slide. Do not take your colonies from a blood agar plate. Blood contains catalase; therefore a false positive reaction would be obtained.
2. Add one or two drops of 3% H2O2 and wait 10-15 seconds to observe.

Catalase Test

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• Interpretation
-- Positive test: immediate bubbling (O2 formed).
-- Negative test: no bubbling.

• Precautions
a. When doing the slide test, always add organism to the slide first and then add the reagent since platinum used in the inoculation needle may produce a false positive result. Nichrome wire does not cause bubbling.
b. H2O2 is very unstable when exposed to light. H2O2 decomposition also increases as temperature increases due to dissolved oxygen. Therefore it is important to keep this reagent in the refrigerator at all times when not in use and to shake before it is used.

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