Is a smear effective for imprinting a skin lesion? Obtaining and processing material for cytological research

smear-imprint

a preparation for microscopy prepared by touching a glass slide to the tissue or organ being examined, followed by drying, fixing and staining.

1. Small medical encyclopedia. - M.: Medical encyclopedia. 1991-96 2. First aid. - M.: Great Russian Encyclopedia. 1994 3. Encyclopedic Dictionary of Medical Terms. - M.: Soviet Encyclopedia. - 1982-1984.

See what a “smear-imprint” is in other dictionaries:

Microscope preparation (see Microscopic preparations), which is prepared from tissues or less often from bacteria growing on solid media. To do this, a piece of tissue is cut out from the organ and its cut is touched one or several times (!) to a glass slide, a smear... ... Dictionary of microbiology

A preparation for microscopy prepared by touching a glass slide to the tissue or organ being examined, followed by drying, fixing and staining... Large medical dictionary

Papanicolaou smear, Papanicolaou test or cytological smear (English Papanicolaou test, Pap test or Pap smear) a test that can be used to identify precancerous or cancerous cells in the vagina and cervix (see Malignant ... ... Wikipedia

Artificial administration to laboratory animals (see) research. ry microorganisms, toxins, research. material that is expected to contain microbes or their toxins. In microbiology Z. e. used to reproduce the disease or its... Dictionary of microbiology

- (histological cytus cell + Greek logos doctrine) is based on the study using a microscope of the structural features of cells, the cellular composition of tissue organs, body fluids of humans and animals in normal conditions and in pathological processes. Ts. and.... ... Medical encyclopedia

- (from ancient Greek: βίος life and ὄψις appearance) a research method in which intravital sampling of cells or tissues from the body is carried out for diagnostic purposes. Biopsy is a mandatory method of confirming the diagnosis when... ... Wikipedia

Biopsy (from the Greek “βίος” life and “όψη” look/appearance) is a research method in which cells or tissues are taken intravitally from the body and then examined microscopically. A biopsy is mandatory... ... Wikipedia

See sign, smallness, consequence and the trace is gone, attack the trace, there is no trace... Dictionary of Russian synonyms and expressions similar in meaning. under. ed. N. Abramova, M.: Russian Dictionaries, 1999. trace mark, imprint, imprint; imprint, print, reflection, ... ... Synonym dictionary

A set of methods for detecting and studying morphological and tinctorial properties in bacteria (microbes) in laboratory samples, pathological material or in samples from the external environment using microscopy. Used to establish the diagnosis of infection... ... Dictionary of microbiology

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In order to determine what condition the patient is in, what disease he is suffering from, the doctor can often use a fingerprint smear. This method is widely used in various fields of medicine. Dermatologists, gynecologists, urologists, oncologists - they all resort to it with varying frequency. A smear examination allows you to quickly determine the pathology and prescribe a competent course of treatment.

A smear-imprint is a medical preparation; it is prepared for use by pressing tissue onto a glass slide. The securely fixed and colored print is examined under a microscope.

The fingerprint smear is used to solve a variety of problems. Most often it is used for:

There is no one general way to take swabs. The method used by the doctor will differ depending on the patient's problem and the purpose of the study.

Biological material from different parts of the body can be used for a smear. If we are talking about diagnosing breast cancer, then the fingerprint material will be taken by the doctor from the patient’s nipples. If an infection is detected, pus will be taken for a smear from the part of the body that was damaged by inflammation. The most commonly used for smears is secretions from abdominal organs.

The problem area can be located not only on the surface of the skin, but also under it. If a doctor encounters a similar situation, he will have to open the skin to obtain prints. To take a smear from hard-to-reach places, special devices are used - tampons, brushes. The discharge remains on them and is then carefully distributed onto a glass slide.

Imprint smear from an ulcer on the genitals

Urologists, venereologists and gynecologists most often have to take prints from the genital organs of their patients. You can usually judge the nature of the infection by scraping the urethra or urethra. If the patient suffers from a minor illness, this may be enough. However, the infection does not always affect the urethra.

Sexually transmitted diseases, that is, sexually acquired diseases, primarily affect not the urethra, but the mucous membrane. This is typical for syphilis and genital herpes, as well as for a number of similar diseases. In a patient experiencing venereal problems, ulcers form on the mucous membrane of the perineum, and a swab-imprint is taken directly from this affected area.

In order to examine a smear taken from an ulcer, specialists use dark-field microscopy. In some cases, it will not be able to determine the disease with absolute accuracy. However, if, as a result of the examination of the prints, treponemes are discovered, the doctor, without doubt, will diagnose the patient with syphilis.

Also, dark-field microscopy allows you to distinguish ulcers that occur with herpes from any other diseases.

Smear impression of the head of the penis

The glans of the penis is often used as an object for a smear in a man. The reason for this may be balanoposthitis, in which the head itself or the foreskin covering it becomes inflamed.

A smear taken from the head allows you to identify a number of problems. It can detect cocci, E. coli, fungal infections and a number of other diseases.

If balanoposthitis, according to the doctor’s suspicion, is of herpetic origin, a fingerprint smear will be taken not only from the head, but also from the vesicles that appear on the skin. If Tzanck cells are found in the vesicles, then the patient suffers from genital herpes. The resulting biological material is stained during examination. This procedure is possible only when vesicles appear.

Imprint smear for cytological examination

A cytological examination of fingerprints is carried out by oncologists to detect atypical cells in the biological material obtained after smears, indicating that the patient is suffering from cancer. In this case, a fingerprint smear is taken from the tumor, which can be located not only on the genitals, but also on other areas of the skin.

During a cytological examination, the entire cellular composition is assessed: the sizes of nuclei and cells are measured.

The technique makes it possible to diagnose not only cancer, but also other processes that are not related to oncology.

Preparing to take a smear

The patient cannot come to the hospital and immediately undergo this procedure. It requires lengthy preparation. If a patient is suspected of having an infection, he should not take antiseptics and antibiotics before taking a smear, as they can distort the clinical picture and even mask the infection.

The patient should not wash the area from which the smear will be taken. If this is necessary for hygienic reasons, you can rinse the area with regular running water without using soap or other cleaning products.

Taking a smear can be hampered by plaque, vesicles and other formations that appear on the skin. In this case, they will be removed before the procedure begins.

How do you study a smear-imprint?

A microscope is required to examine the smear taken. The slide on which the biological material is placed must be absolutely clean and dry.

The drug can be fixed using various methods. Typically, heat, 95% ethyl, methyl or sublimate alcohol, and acetic acid vapor are used for fixation. The method chosen depends on the laboratory in which the research will be carried out. Today, specially developed preparations, which are a mixture of a number of chemicals, are actively used for fixation.

The fixative is critical to the effectiveness of the entire procedure. If a specialist uses an insufficiently high-quality solution, it will have a detrimental effect on the structure of the cells in the smear and will likely give a false negative result. Based on the results of the analysis, the patient will be informed that he is absolutely healthy, but in reality this is not the case. Only a high-quality fixation solution can preserve the correct location of all cells of the smear and identify bacterial formations among them.

The fixed material is subjected to staining, which can be indicative or differential.

Authors): Jan Rybniček, MVDr, Diplomate European College of Veterinary Dermatology
Organization(s): Dermatology and Dermatopathology Service, Czech Republic
Magazine: №2 - 2013

Most skin diseases have a similar clinical picture, but nine main groups of characteristic dermatological disorders can be distinguished:

– alopecia

– formation of scales/crusts/seborrhea

– macules/papules/opening pustules

– nodes/tumors

– changes in pigmentation

Once the clinician begins to classify each patient into one of the categories listed above, it will become much easier for him to formulate a list of differential diagnoses and develop a diagnostic plan. It must be remembered that the patient may have concomitant diseases at the current time that make it difficult to make the main diagnosis .

A quick diagnosis often poses significant difficulties for a practicing physician, so further we will talk about available laboratory research methods that require minimal time and money. The simplest and fastest diagnostic tests used in practice:

– Skin scrapings

– Trichoscopy

– Combing with a comb or brush

– Scotch test

– Test for dermatophytes

– Skin cytology

Skin scrapings

This study must be carried out in all cases! To carry out skin scrapings, the following equipment and materials are needed: mineral oil, glass slides, scalpel blade or spatula (curette), microscope.

Examination in the rays of a Wood's lamp is extremely nonspecific, however, when fluorescence is detected, the affected hairs are plucked out and subjected to further examination (microscopy after treating the drug with an alkali solution, culture to identify a culture of dermatophytes). A 10% KOH solution is applied to a glass slide, the removed wool is placed, heated over a flame, and microscopy is carried out, first covered with a cover glass, first under low magnification (10x objective), then under high magnification (40x objective). Chlorolactophenol staining can be used for better visualization.

Scotch test

To obtain material for cytology, it is necessary to cut the hair short from the selected area, press the strip of tape tightly, then it is dipped one by one in dye solutions, glued to a glass slide, and microscopy is performed. This method easily identifies yeast fungi of the genus Malassezia in the shape of a “matryoshka”, bacteria of the genus Simonsiella(indicates the entry of saliva during licking), etc.

Trichoscopy

During microscopy, attention is paid to the structure and pigmentation of the hair shafts, the ends of the hair are examined, and the trichogram is assessed (the ratio of hair in different phases of the follicle cycle: anagen/telogen). Anagen is the growth stage, the root of the removed hair is soft and takes on a rounded shape (“umbrella handles”). Catagen is an intermediate stage, hair growth stops, the root has the shape of a brush surrounded by a glassy membrane. Telogen is the resting stage of the hair follicle, the hair root loses pigment, tapers towards the end in the shape of an “art brush”, and takes on the appearance of a “spear”. There are primary and secondary hairs (normally). Loss of primary hair may be one of the diagnostic criteria for some alopecias (eg, alopecia X). Primary hair shafts are larger in diameter, and the medulla is always thicker than the cortex. Secondary hair is smaller in diameter, often wavy, the medulla is thinner than the cortex.

There are no strict standards for Trichogramma, however there are observations describing an anagen/telogen ratio of 1:9 in winter and 1:1 in summer in some dogs. There are breed characteristics - in dogs with constantly growing hair (for example, poodles), follicles in the anagen stage predominate, regardless of the time of year, and in dogs of northern breeds, pronounced telogenization of the follicles can be observed.

The presence of 100% of hair in the telogen stage in most cases is not the norm; it may be due to improper sampling, hormonal disorders, alopecia X, telogen effluvium. Hormonal factors influence the hair follicle cycle and are accompanied by telogenization; due to a delay in the hair follicle cycle, the hair gradually undergoes the aging process (black wool becomes reddish, red hair becomes discolored, the ends may split). The primary hair disappears, and the retained undercoat has the appearance of puppy hair. Endocrine causes of such alopecia can be hypothyroidism, hyperestrogenism, hypercortisolism. A non-endocrine cause is the synchronization of all telogen hairs caused by severe illness or stress. As soon as the hair resumes its growth, remission occurs after 1-2 months. Alopecia X is a disease with an unclear etiology.

Structural disorders of the hair roots occur due to follicular dystrophy or dysplasia. In rare cases, this may be due to damage from pigment granules. Hair roots are also damaged by alopecia areata and exposure to cytotoxic drugs (rarely).

Pay attention to the ends of the hair: normally they have a pointed shape, but with alopecia due to licking, signs of trauma to the ends of the hair are observed (they may be broken off). Hair ends may become damaged due to increased fragility caused by structural changes. For example, the appearance of forked hair ends due to trichophytosis in Golden Retrievers has been noted. Pathological changes in the ends of the hair can be the result of extremely aggressive washing and combing of the coat, as well as mechanical damage in areas of increased friction.

When assessing the hair shaft, dermatophyte spores may be detected. When performed correctly, trichoscopy gives a positive result in 60-70% of cases of dermatophytosis. It is preferable to examine hair that fluoresces under a Wood's lamp. The structure of the hair shaft is disrupted not only with dermatophytosis, but also in a number of other cases, for example, chemotherapy (rare), mechanical and chemical trauma (aggressive care), lack of nutrients, hereditary diseases (nodular trichorrhexis, etc.). Characteristic changes in the structure of the hair shaft are observed in medullary trichomalacia of German shepherds, which manifests itself in adult dogs. The coat appears trimmed on the body, tail, and shoulder-scapular area. During trichoscopy, longitudinal cracks and breaks are observed in the hair shafts. Characteristic changes are observed in alopecia associated with impaired melanin production - alopecia of “weakened” color and follicular dysplasia of black hair. Blue and light brown dogs are susceptible to “bleaching alopecia.” With this disease, large pigment granules are found in hair with weakened color, which cause increased hair fragility, which leads to alopecia. Black hair follicular dysplasia occurs in bi- and tri-colored dogs, affecting only the black hair follicles. Puppies are born normal, but subsequently the areas of dark hair gradually undergo alopecia. Trichoscopy reveals abnormal clumps of melanin in the hair shaft.

Trichoscopy can easily detect keratin aggregates in the form of casts of hair follicles on the hair shaft. They arise as a result of keratinization defects (primary seborrhea of ​​cocker spaniels, dermatosis due to vitamin A deficiency, adenitis of the sebaceous glands, leishmaniasis, demodicosis), as well as other follicular diseases, for example, bacterial folliculitis.

Skin cytology

Skin cytology is a fast, non-invasive method that has great diagnostic value. This type of study is especially valuable for the diagnosis of nodular, exudative, pustular lesions, with the formation of crusts and seborrhea, as well as with otitis media. For nodular lesions, it is preferable to obtain material by fine-needle aspiration using a 10 ml syringe with 21G, 24G needles. For oozing lesions, fingerprint smears can be examined. Also, adhesive tape preparations and, of course, deep scrapings are subjected to cytological examination. The preparations are dried in air; fixation with methanol can be used. I use a modified Wright stain (Diff-Qick), and laboratories also perform staining with special dyes and immunocytochemistry. The preparations are viewed sequentially using 4x, 10x, 40x, 100x lenses (with immersion oil).

In scrapings from normal skin, corneocytes predominate; other keratinocytes and single fibroblasts are also present. When performing a cytological examination of material from inflamed areas, attention is paid to the following cells:

– Neutrophils (degenerative and non-degenerative);

– Macrophages;

– Lymphocytes/plasma cells;

– Eosinophils and mast cells;

– Acantholytic cells (rounded keratinocytes, devoid of processes);

– Neoplastic cells.

Microorganisms (bacillus, cocci, Malassezia, other fungi, Leishmania, etc.) can also be detected.

I use the following main criteria for cytological differential diagnosis:

– Bacteria and epithelial cells – bacterial overgrowth;

– Degenerative neutrophils and bacteria – pyoderma;

– Non-degenerative neutrophils in large numbers + acantholytic cells – pemphigus foliaceus, erythematous pemphigus, etc. (additionally + histopathology);

– Neutrophils + macrophages – pyogranuloma/granuloma (fungal, atypical bacterial infection);

– Lymphocytes, plasma cells – chronic inflammation, plasmacytic pododermatitis, lymphoma (additionally + lymphoblasts);

– Malassezia sp. and epithelial cells – Malassezia dermatitis.

When assessing cell morphology, it is customary to evaluate the following signs of malignancy:

– Cytoplasmic criteria (anisocytosis, macrocytosis, different color intensity (degree of basophilia), atypical inclusions/vacuolization, absence of specific granules, large nuclear-cytoplasmic ratio, as well as the presence in the preparation of cells that differ significantly in this parameter);

– Nuclear criteria (anisokaryosis, macrokaryosis, changes in nuclear shape, sparse chromatin or hyperchromasia, anisochromasia, high mitotic index, irregular mitoses, multiple nucleoli, giant nucleoli, pathological shape of nucleoli).

When examining cytological preparations of nodular lesions, the following questions must be answered:

– Inflammation/tumor;

– Neoplasia – benign/malignant;

– Source of neoplasia;

– Tissue origin: epithelial, mesenchymal, hematopoietic (round cell) tumors.

The most common cytological differential diagnoses of tumor skin lesions in practice are:

– Epithelial tumors (keratoacanthoma, squamous cell carcinoma and others);

– Mesenchymal tumors (fibrosarcoma, melanoma, etc.);

– Round cell tumors (mastocytoma, histiocytoma, lymphoma);

Due to the increasing availability of histopathology in recent years, the ability to diagnose various dermatological pathologies has significantly increased. Despite this, it is still quite rare to resort to taking histobiopsy specimens due to the possibility of obtaining uninformative results. Therefore, further information is devoted to assessing the need to take a skin histobiopsy, the correct selection of the skin area for histological examination, the correct sample collection, the choice of a pathologist and the interpretation of the histological report. Only if all these conditions are met can an accurate diagnosis be obtained.

1) Histobiopsy is not routinely used for diagnosis in dogs with chronic pruritus. In most cases, the results of a biopsy will not provide the same information that a dermatologist can obtain during a clinical examination.

2) Skin histobiopsy may be useful in the following cases:

– Alopecia, when it is not possible to make a diagnosis using “quick” diagnostic methods (i.e. trichoscopy, skin scrapings, cytology...)

– Peeling when diagnosis cannot be made using “quick” tests

– Spots, papules, ruptured pustules when diagnosis cannot be made using “quick” tests

– Non-healing erosions, ulcers, fistulas

– Long-term nodes, compactions, tumors

– Depigmentation and unusual hyperpigmentation

– Suspicion of autoimmune diseases

– Suspicion of skin diseases, for which confirmation of the diagnosis is only possible histologically (for example, adenitis of the sebaceous glands)

– Cases when a life-threatening diagnosis is suspected (dermatomyositis, etc.)

– Cases where there is no response to completely rational treatment

How to obtain high-quality skin histobioptates

To obtain a clear, correct histological diagnosis, the following steps should be followed:

1) Selecting a site for biopsy

Appropriate specimens must be presented to the pathologist, so the clinician must take care to select the appropriate skin site. In most cases, at least 3-5 skin samples must be taken from one patient to more likely obtain a correct diagnosis. The choice of biopsy site depends on the type of lesion found:

– Alopecia: Biopsies should be obtained from an area of ​​completely bald skin (preferably 2), at the border of normal skin and an area of ​​alopecia (1) and from skin covered with normal hair (if any) so that the pathologist can compare physiological hair follicles and follicles from the area of ​​the pathological process of this patient.

– Scaly lesions (“seborrheic” skin): 2-3 biopsies are taken from the affected area and a sample of normal skin (1) so that it is possible to compare the epidermis, especially the stratum corneum in these areas. A biopsy needle can be used to obtain samples.

– Macules, papules, pustules: The biopsy should obtain samples from at least three sites of primary lesions, which should be located in the center of the biopsy. A biopsy needle can be used to obtain samples.

– Ulcerated areas: This is the most difficult type of lesion to biopsy. The main mistake is sending a biopsy from the center of the ulcer to the pathologist and then receiving the answer “ulcer” without any specific diagnosis. In a situation where the material is obtained from the center of a large ulcer, the histological picture will be characteristic of an infectious process. It is also necessary to take several samples so that the pathologist can compare the processes occurring in the ulcer and in normal skin. If small ulcers are present, the entire area, including the rim of healthy tissue, can be sent for examination.

– Fistulas: Usually the pathological process is located deep in the dermis, and often under the skin, so a very deep biopsy is needed to determine the nature of the pathological process. In such cases, a wedge-shaped biopsy is excised using a scalpel blade; with this type of lesion, it is impossible to obtain an informative sample using a biopsy needle. At the same time, it makes sense to send the biopsy material for bacteriological and mycological research.

– Seals and components: again, surgical excision of the entire node with adjacent normal tissue is the treatment of choice. According to established rules, this procedure can be performed only after mastocytoma has been excluded by performing fine needle aspiration and cytological examination.

– Depigmentation: histobioptates are obtained from completely depigmented areas (minimum 2), from the border zone between normal and depigmented skin (1), as well as one sample from normal skin.

– Hyperpigmentation: histobioptates are obtained from pigmented areas (minimum 2), as well as one sample from normal skin.

2) Interpretation of the histological report

Typically, the dermatohistopathologist issues a report that should contain the following sections:

1. Description of the tissue sample

2. Morphological diagnosis

3. Etiological diagnosis (if it is possible to determine the cause)

4. Comments

The histological description represents the information that the pathologist sees in the preparations. The histological description contains valuable information using a variety of terms, which is of great importance in diagnosis. However, considerable experience is required to explain these observed changes, so the clinician should not always trust the interpretation of the pathologist's report. Knowledge of basic terminology is essential for correct interpretation of laboratory findings. Unfortunately, most clinicians do not read the narrative at all.

The morphological diagnosis is a one-sentence summary of the pathological changes in the histological specimen (something like: marked hyperplasia with crusting and perivascular eosinophilic infiltration with scabies mites in the stratum corneum). Morphological diagnosis is based on the terms used in the description of the histopathological specimen.

Etiological diagnosis is possible when histopathology is specific for a disease. For example, in the case described above, the etiological diagnosis is “sarcoptosis.” If an etiological diagnosis cannot be established, the pathologist offers a list of differential diagnoses and, possibly, recommendations for further diagnosis.

The clinician should never blindly rely on the report; it is important that the clinician connects the clinical picture and histopathological information together. There are a large number of diseases that can be diagnosed histologically, but many of them can be suspected based on characteristic features.

The most common histopathological findings will be given below.

Epidermal disorders

2. Hyperplasia- this is a nonspecific response of the epidermis to chronic inflammation or injury, observed in many inflammatory processes accompanied by itching.

3. Subcorneal and intraepidermal pustules– usually found in superficial pyoderma, as well as in pemphigus foliaceus and some rare sterile pustular diseases.

4. Subepidermal pustules and vesicles- this is a very rare symptom, found in hereditary and autoimmune diseases, for example, in pemphigus vulgaris.

5. Necrosis and ulcers– usually seen in burns, contact dermatitis, pyotraumatic dermatitis (“hot spots”), feline head and neck pruritus syndrome, feline eosinophilic complex. More rare causes are hepatocutaneous syndrome, erythema multiforme/toxic epidermal necrolysis, some drug reactions, feline viral diseases (cowpox virus, herpes virus), thymoma in cats. Ulcers may be secondary lesions due to serious skin diseases such as vasculitis.

Dermal lesions

1. Superficial lesions of the dermis– quite rare, can be observed with pyoderma of the mucocutaneous areas, lupus, erythema multiforme, epitheliotropic lymphoma, thymoma (in cats), dermatomyositis.

3. Folliculitis/furunculosis– in most cases, indicates a skin lesion of an infectious nature, caused, for example, by staphylococci, dermatophytes or demodexes. Other causes may be eosinophilic furunculosis, pemphigus foliaceus, and epitheliotropic lymphoma.

5. Panniculitis– this sign can be observed with deep subcutaneous bacterial, mycobacterial and fungal infections, with sterile nodular, idiopathic panniculitis, pansteatitis, lupus, as well as with local reactions to injections and injuries. May be a consequence of pancreatitis.

6. Vasculitis- this sign indicates direct damage to the blood vessels. Vasculitis can be present in a wide variety of conditions, such as septic vasculitis, immune-mediated diseases, and due to drug exposure.

7. Atrophy– observed in endocrine diseases, some specific alopecias, such as seasonal alopecia of the lateral surface of the body, paraneoplastic syndrome and ischemic dermatopathy.

To summarize, we can say that if in animals with signs of alopecia, peeling and crusts, macules, papules, pustules, I cannot establish a diagnosis by performing all of the above quick tests, I resort to performing histobiopsy. It must be remembered that in some cases histological examination is not very informative (for example, in animals with chronic itching), so I would like to emphasize the extreme importance of rapid tests in these patients, as well as the need for clinical assessment and careful analysis of anamnestic data by a veterinary specialist.

Literature

1. Cowell R.L., Tyler R.D., Meinkoth J.H., DeNicola D.B. Diagnostic Cytology and Hematology of the Dog and Cat, 3rd ed., Mosby Elsevier, 2008.

2. Raskin R.E., Meyer D.J. Canine and Feline Cytology, Saunders Elsevier, 2nd ed., 2010.

3. Mc. Cullough S., Brinson J. Clinical Techniques in Small Animal Practice, Vol.14, No.4, November, 1999.

4. Taylor S.M. Small Animal Clinical Techniques, Saunders Elsevier, 2010.

Preparation of the colored preparation consists of the following steps:

1) preparation of smears;

2) drying the smear;

3) fixation of the smear;

smear coloring.

To prepare the preparation, a drop of water or saline is applied to a fat-free glass slide, into which the test material is inserted in a loop and distributed in a thin, uniform layer over the glass over an area of ​​approximately 1 cm 2 . If the material under study is in a liquid medium, then it is directly applied with a loop to a glass slide and a smear is prepared. The smears are dried in air or in a stream of warm air over the flame of an alcohol lamp, without allowing the drop to boil.

To fix the smear, a glass slide (smear up) is slowly passed through the flame of an alcohol lamp 3-4 times. Microorganisms die when fixed, are tightly attached to the surface of the glass and are not washed off during further processing. Longer heating may cause deformation of cellular structures.

When fixing with chemicals, chromium compounds, formalin, osmic acid, and acetone are used. One of the common fixation methods is treating the preparation with methyl or ethyl alcohol, or Nikiforov’s mixture (equal volumes of ethyl alcohol and ether). In this case, the drug is immersed for 5-20 minutes. into the fixing liquid.

Methods of staining smears and dyes used in microbiology.

Exist simple and complex painting methods . For simple coloring, use one of the dyes, for example, aqueous fuchsin (1-2 minutes), methylene blue (3-5 minutes). When staining a smear, the drug is placed on a drug holder. A few drops of dye are applied to the smear. After the staining time has expired, the preparation is washed with water, excess water is removed with filter paper, air-dried and microscopically examined.

For complex staining, certain dyes differing in chemical composition and color are sequentially applied to the preparation. This makes it possible to identify certain cell structures and differentiate some types of microorganisms from others. These are the Gram staining methods, Ziehl-Neelsen staining, and spore staining using the Ozheshka method.

Dyes that release hydrogen ions upon dissociation, giving the dye an acidic character, are called sour. They color (in the form of an anion) substances of a basic nature. Dyes that release hydroxyl ions upon dissociation - main ones.

In microbiological practice, acidic and basic dyes are used in the form of salts, since they are capable of reacting with acids and bases. Basic dyes are often used in the form of salts of hydrochloric, less often acetic and sulfuric acids; acid dyes - in the form of sodium or potassium salts.

Basic nature dyes

Red Purple

neutral red gentian violet

pyronine crystalline violet

safranin methyl violet

fuchsin methyl green

hematoxylin malachite green

Blue Brown

Victoria Vezivin

methylene blue chrysoidin.

Green Black

Janus green indulin

Dyes of acidic nature

Red and pink Black Yellow

sour fuchsin nigrosin Congo

erythrosine picric acid

fluorescein

The intensity of the dye's coloring ability depends on the pH of the medium: basic dyes color an object more intensely the more alkaline the environment, acidic dyes the more acidic it is.

Dyes can be divided into: positive and negative. Positive dyes directly stain microbial cells. They stain cells at room temperature for 30-60 seconds. Negative dyes color the space surrounding the cells of microorganisms. As a result, the cells appear as silhouettes against the background of the dye.

Simple painting method

Apply a few drops of an aqueous solution of fuchsin to the prepared and fixed smear. Paint for 1-2 minutes. Wash off the paint with water, blot the preparation with filter paper, air dry and microscope.

Gram staining.

Apply a carbolic-alcohol solution of gentian violet to the fixed smear through a strip of filter paper. After 1–2 minutes, remove the paper and drain the dye.

Apply Lugol's solution for 1–2 minutes.

Decolorize with ethyl alcohol for 30–60 s until the violet dye structures stop coming off.

Rinse with water.

5. Finish with an aqueous solution of fuchsin for 1-2 minutes, rinse with water, dry with filter paper and air, and microscope.

Gram-positive bacteria are stained dark purple, while gram-negative bacteria are stained red.

The attitude of bacteria to Gram staining is determined by their ability to retain the complex of gentian violet with iodine formed during the staining process. This depends on differences in the chemical composition and permeability of the cell wall of gram-positive and gram-negative bacteria. Peptidoglycan is multilayered and teichoic acids are associated with it.

In gram-negative bacteria, the peptidoglycan is single-layered, there is an outer membrane, which includes phospholipids, lipoproteins, proteins and complex mepopolysaccharide (LPS). The entire outer membrane is permeated by proteins - porins, which ensure the diffusion of various compounds. Thus, optimal conditions are created for gram-positive bacteria for strong fixation of the dye and resistance to bleaching with alcohol.

Gram-positive bacteria include staphylococci, streptococci, corynebacterium diphtheria, mycobacterium tuberculosis, etc., and gram-negative bacteria include gonococci, meningococci, Escherichia coli, etc. Some types of bacteria can be Gram stained variably, depending on age, cultivation characteristics and other factors affecting on the structure of the cell wall. Therefore, for coloring you should always take young, 1-day old cultures.

The main mistake made when staining according to Gram is “re-bleaching” the smear with ethyl alcohol. In this case, Gram-positive bacteria can lose their original gentian violet color and acquire a red color (characteristic of gram-negative bacteria) as a result of subsequent staining of the smear with fuchsin; gram-negative bacteria, in turn, can retain the blue-violet color of gentian violet. For proper coloring, the bleaching technique must be strictly followed.

Normally, a woman without lactation has no discharge from the breast. The fluid (exudate) flowing from the mammary gland is analyzed by taking fingerprint smears. The contents of cysts and skin ulcers are also examined.

Why is the test taken - breast smears?

Using this method, the disease that caused the appearance of discharge from the mammary glands is determined. A cytological analysis of the fluid is performed to determine the cellular composition. In case of malignant tumors, atypical (cancerous) cells are detected in the smear. With the help of fingerprint smears they diagnose:

• breast cancer
• mastopathy and mastitis;
• galactorrhea - secretion of milk from their glands, associated with hormonal imbalance;
• mammary tuberculosis;
• benign formations;
• blockage and expansion of ducts;
• contents of cysts and wound surfaces

How is a breast examination performed?

The discharge from the nipples and wounds on the skin is applied to a piece of glass, dried and sent to the laboratory.

To examine the contents of the cysts, a puncture is performed to collect fluid. The injection is done with a thin needle, so no anesthesia is required. After the test is taken, the patient goes home. A woman applies ice to the puncture site at home. The point is applied to glass and sent for testing.

The procedure is performed on an outpatient basis and does not require hospitalization. Before the procedure, you should not take drugs that change blood clotting. On the day of the study, it is prohibited to apply creams, sprays, or deodorants to the chest and armpit area.

Breast smear results

In the laboratory, the material is stained and examined under a microscope:

• in case of injuries and non-malignant diseases of the mammary glands, blood and leukocytes are present in the fluid. Benign tumor cells may be detected. In this case, the woman is examined additionally;
• in case of breast cancer, atypical (cancerous) cells are found in the discharge - large, irregularly shaped with several small “nucleoli”. The accuracy of the analysis is close to 100%. During the study, the type of malignant tumor is determined;
• In the initial stages of breast cancer, altered cells in the smear are not always detected. In 15%, the oncological process is detected by other methods - Doppler ultrasound, MRI. If the result is questionable, the woman will be examined again.

Taking fingerprint smears from the mammary gland is a low-traumatic and informative method for diagnosing cancer and other pathologies of the mammary gland.