Wet Mount Microscopy: Introduction, Principle, Preparation, Result Interpr

Wet Mount Microscopy: Introduction, Principle, Preparation, Result Interpretation, Limitations and Related Videos

Introduction of Wet Mount Microscopy

Wet Mount Microscopy is a very simple technique that is applied widely in clinical laboratories. e.g. Saline wet mount and iodine wet mount for stool examination, potassium hydroxide (KOH) mount of skin, nails, hairs for fungal elements, vaginal wet mount for vaginal yeast infection ( candidiasis), trichomoniasis, and bacterial vaginosis. Saline wet mount microscopy of the stool or stool wet mount microscopy is the simplest and basic method for the study of feces and is applicable in every medical laboratory even in a small setup. Saline wet mount microscopy uses for the following purposes-

To observe live trophozoites ( e.g. Entamoeba histolytica/dispar, Girdia lablia, Trichomonas, etc.) and larvae of parasites ( e.g. Strongyloides stercoralis) are motile except inactive forms.
To find out eggs, cysts, oocysts of different parasites ( Helminths, protozoa, and coccidian parasites).
To determine the presence of leukocytes and erythrocytes in a fecal smear.
It also gives clues towards the motility of bacteria (Shigella non-motile causing bacillary dysentery whereas Vibrio cholerae shows darting motility which is causative agent cholera).
It also remarks the presence of fungal elements ( yeast cells, hyphae, or fungal spores).
It also visualizes the presence of non-parasitic structures like Charcot Leyden crystal, muscle fibers, fat globules, starch cells, vegetable fibers, hair, etc.
The growth of yeasts and bacteria on culture media may be differentiated by this simple technique.
The shape of bacteria, cocci, or bacilli can be differentiated from the colony of the culture plate by saline wet mount microscopy.

Principle of the Saline Wet Mount Microscopy

Saline wet mount preparation for stool uses analyzing a stool specimen in coprology (study of feces). It utilizes a physiological saline solution (0.85% NaCl ) as an isotonic media to maintain the cellular structure of the various organisms as well as our cells that are found in stool and other stools too.

Requirements for Saline Wet Mount Microscopy

Physiological saline ( 0.85% NaCl)
Specimen: stool
Sterile bamboo sticks or low cone on the end of a wooden applicator stick
Clean and grease-free slides and
Cove slips(22- by 22-mm)
Microscope
Gloves

Saline wet mount of stool Preparation

First, wear the groves.
Take a clean and grease-free slide.
Add one drop of physiological saline and then add a stool equivalent to a match stick head (2 mg) with the help of a stick.
Mix it properly and apply a coverslip over a uniform suspension without creating bubbles.
Note: If a fresh stool specimen is received and if blood and mucus are present, the specimen should be examined as a direct mount making sure to sample the bloody areas.
Examine the entire 22- by 22-mm coverslip systematically with the low power objective (10X ) and low light intensity.
If any suspicious objects encounter, examine with the high dry objective (40X).

Result Interpretation of Feces Saline Wet Mount Microscopy

Presence of active trophozoite/s: Motile retractile bodies

Cyst, oocyst, egg, inactive trophozite/s, larvae: Retractile bodies and finally focus at high dry power field

Keynotes on Saline Wet Mount Microscopy

There is little difference between normal and physiological saline. Physiological saline is 0.85% NaCl whereas normal saline is 0.9% NaCl.
Gram’s iodine is not applicable for staining parasitic organisms and for this D’Antoni’s iodine uses.
Oil immersion examination is also preferred in parasitology for the permanent stained smear of parasites.
Intestinal protozoa can not conform on the basis of a wet mount alone and thus permanent stained smears require to confirm the specific identification of suspected organisms.

Limitations of Saline Wet Mount Microscopy

Due to the lack of stain, it is difficult to get morphological details.
Inappropriate preparation of the smear may hide parasites.
Improper adjustment of the microscope in relation to the objective may create problems.

Wet Mount Microscopy Related Videos-

Heavy load of parasites in stool|| Stool microscopic examination|| Trichomonas hominis in a saline wet mount of feces as shown below-

Trichuris trichura or whipworm under saline wet mount at 40X objective under the microscope –
Features: Barrel shape
Mucus thread at each pole as shown in the video

Egg of Taenia species
They are spherical, brown in color (bile stained), and measure 30-40 µm in diameter.
They are surrounded by embryophore which is brown, thick-walled, and radially striated.
Inside embryophore, the hexacanth embryo (oncosphere) presents three pairs of hooklets.
They do not float in a saturated solution of common salt (brine solution).
They are viable for 8 weeks.

Roundworm or Ascaris lumbicoides egg under the microscope in saline preparation
egg-infertile
Finding of eggs
In stool:
direct microscopic examination of a saline emulsion of the stool
Concentration methods may be used.
Note:
The fertilized egg floats in a salt solution
Unfertilized eggs do not float

Hymenolypsis (now called Vampirolepsis) egg in a saline wet mount of stool: showing hooklets egg of Hymenolepis liberated in feces by the gradual disintegration of terminal segments

Spherical or oval in shape, 30-45 µm

Two distinct membranes

Outer membranes are thin and colorless

Inner embryophore encloses an oncosphere with 3 pairs of hooklets

Space between two membranes –filled with yolk granules and polar filaments emanating from little knobs at either end of embryophore.

Egg of hookworm ( Ancylostoma duodenale or Necator americanus) in saline preparation of stool under the microscope
Egg features-
Shape: oval or elliptical with flattened poles( one pole more often flattened than other), size: 65 X 40 um, color: colorless ( no bile stain), dark brown as stained with iodine. Shell: very thin transparent hyaline shell membrane, appears as a black line and contains: segmented ovum with 4 blastomeres, has a clear space between eggshell and segmented ovum. Float in saturated NaCl. Type: A( fresh stool) : 4 ,8, 16 grey granular cell clear blastomeres. Type: B(a few hours old): a uniform mass of many grey granular cells. Type: C( 12-48 hr): the whole egg is filled with larva, embryonated.

Enterobious vermicularis ( common manes pinworm or threadworm or seatworm) – eggs and some are with larva in a saline wet mount of feces –
Shape: oval, planoconvex.

Size : 50-60μm x 20-30μm.

Surrounded by double-layered eggshell

Embryonated when passed fresh; contains a tadpole larva inside.

Entamoeba coli Cyst with 8 nuclei in iodine wet mount as shown below-

Giardia lamblia cysts in iodine wet mount as shown below ( look at center)-

Entamoeba histolytica (Amoeba) trophozoites and cyst in LPCB preparation as shown below-

Blastocystis hominis cyst in Sargeaunt stained slide under the microscope as shown below-

Oocyst of Cyclospora cayetanensis ( coccidian parasite) in a saline wet mount of stool under the microscope as shown below-

A very simple Saline wet mount technique helps you to identify yeast cells of candida from bacteria-

Bacteria and Fungus in saline wet mount Microscopy at Various magnifications 400X, 800X and 1600X

Introduction of KOH Wet Mount

The KOH Wet Mount is very important for presumptive diagnosis of the type of fungal infections whether ringworm, aspergillosis, dermatomycosis, blastomycosis, mucormycosis, otomycosis, and so on. It is also helpful in the selection of appropriate culture media for the isolation of etiological fungal agents.

Principle of KOH Wet Mount Preparation

As you know, potassium hydroxide (KOH) is a strong alkali. When specimens such as pus, skin, hair, nails, or sputum mix with, it softens, digests, and clears the tissues i.e. keratin present in the skin surrounding the fungi so that the fungal elements( yeast, a cell with pseudohyphae, budding, hyphae, granules, conidia, etc.) of fungi can be seen under a microscope.

Requirements for KOH Wet Mount Preparation

1. Equipment

Microscope

2. Reagent and laboratory wares

Glass Petri dishes
clean and grease-free glass slide
Cove slip
Straight wire or bent wire
Needle
Bunsen burner
20% KOH

3. Specimen

It may vary according to the site of infections such as pus from draining sinus, aspirate from nasal sinuses, respiratory specimen, skin scrapings, nail clipping, hair, corneal scraping, material from ear discharge, etc.

Procedure of KOH Wet Mount Preparation

Emulsify the specimen in a drop of 20% KOH on a glass slide with the help of inoculating loop. To assist clearing, hairs should not be more than 5 mm long, and skin scales, crusts, and nail snips should not be more than 2 mm across.
Apply gentle heat by passing the slide over a Bunsen burner 3-4 times in case of 10% KOH or hard specimens like nail clipping or hair. (Note: This step can omit by using a high concentration of KOH or KOH with DMSO or treating the specimen with KOH for a longer duration.)
Cover the smear with the coverslip and leave for 5-10 minutes. (generally, bur when using nail clipping or hair, there is a need for a longer duration.)
As soon as the specimen has cleared, examine the preparation microscopically focusing the 10X and finally observation using 40X objectives with the condenser iris diaphragm closed sufficiently to give a good contrast. If you are using too intense a light source the contrast will not be adequate and the unstained fungi will not be seen. Examine the preparation carefully for the demonstration of shining fungal elements.

Quality control (QC)

It can maintain using the following points-

The right concentration of 20% KOH should be prepared.
Emulsification of specimen should be homogenous in potassium hydroxide (KOH) preparation.

Observation

Observe fungal elements in microscopy of the clinical specimens.

Result and Interpretation of KOH Wet Mount

If there is the presence of any fungal elements either yeast cells, cells with pseudohyphae, budding, septate hyphae, aseptate hyphae, branching hyphae, conidia, or granules, etc. during the examination, KOH mount is positive i.e. fungal elements seen.

No fungal elements were seen during in microscopy of the clinical specimen, KOH preparation is negative.

Interpretation of results should do by critical analysis of the type, size, and color of the fungal elements that will be different for different fungi. A fungal culture is necessary for the isolation of etiological fungal agents.

Uses of KOH Wet Mount Preparation

The uses of KOH wet mount preparation are as follows-

It is used for the rapid detection of fungal elements in clinical specimens, as it clears the specimen making fungal elements more visible during direct microscopic examination.
It is very useful for the presumptive diagnosis of type fungal infections.
The recommendation of KOH wet mount preparation is very useful in the following suspected clinical conditions as shown in this table.

Keynotes on KOH Mount

Potassium hydroxide(KOH) reagent is highly corrosive therefore handle it with great care.
Experience is necessary for reporting because background artifacts are often confusing.
Clearing off some specimens such as nail clipping, hair may require an extended time.

Introduction of Vaginal Wet Mount

The vaginal wet mount also called the vaginitis test is very useful for diagnosis vaginal infections that could be causing vaginitis. Vaginitis encompasses a variety of disorders that cause infection or inflammation of the vagina. The causative agents of vaginitis can include bacteria, yeast, viruses, and parasites. Non-infectious vaginitis may also occur due to estrogen imbalance (low level) that causes vaginal dryness as well as a reaction to vaginal products such as soap, bath oils, spermicidal jelly, or douches.

The most common pathogens are as follows-

Bacteria: Gardnarella is a commonly found bacteria in the vagina. Overgrowth results in bacterial vaginosis, Streptococcus or Staphylococcus can also be present but usually do not result in an infection. A bacterial infection can cause a grayish-white discharge with a fishy odor.

Fungi: Candida albicans cause genital itching and a thick, white vaginal discharge with a cottage cheese-like texture.
Viruses: Human papillomavirus (HPV)
Parasites: Trichomonas vaginalis, Pinworms, scabies, and lice can cause inflammation of the vulva and vagina.
Vaginitis is also a sexually transmitted infection (STI).

Symptoms of vaginitis

Common symptoms of vaginitis are as follows-

vaginal discharge that may have an odor
itching or swelling on the outside of the vagina
burning during urination
pain or discomfort during intercourse

Principle of wet mount

Saline wet mount preparation for vaginal smear analyzing a vaginal discharge specimen for diagnosis the causative agents of vaginitis. It utilizes a physiological saline solution (0.85% NaCl ) as an isotonic media to maintain the cellular structure of the various organisms as well as patient cells that are found in vaginal discharge.

Vaginal Wet Mount or Vaginal smear Preparation

A wet mount of vaginal smear also called wet preparation uses to find out the causative agents of vaginal infections that don’t affect the urinary tract. A clinician ( gynecologist) will have you lie down on an exam table with your feet in stirrups, like at a regular gynecologic exam. She/he will insert a speculum into the vagina to help see the area. After that, a sterile cotton swab is inserted into the vagina to obtain a sample of vaginal discharge. The clinician will transfer the sample onto a slide or leaving on the swab. The slide is sent to the microbiology section to check the causative agent of vaginitis.

Requirements for the vaginal wet mount test procedure

Test slides or vaginal discharge on the swab
Physiological saline or normal saline
Microscope
Coverslips
Sterile Wooden Applicator Stick
Gloves

Wet mount procedure

First, wear gloves.
Take a clean and grease-free slide ( no need in case of slides provided by a clinician).
Place s sample of the vaginal discharge on a glass slide.
Add one drop of physiological or normal saline
Mix it properly with a sterile wooden applicator stick and apply a coverslip over a uniform suspension without creating bubbles.
Examine the entire 22- by 22-mm coverslip systematically with the low power objective (10X ) and low light intensity.
If any suspicious objects encounter, examine with the high dry objective (40X).

Observation of Vaginal smear

Look at under a microscope for bacteria, yeast cells, Trichomonas, pus cells ( dead white blood cell), and clue cells that is an indicator of bacterial vaginosis.

Result Interpretation of vaginal smear or vaginal wet mount

Bacteria: The organisms which have a size of this range 0.2 – 2.0 × 1 – 10 µm and shape of normally round or rod.

Trichomonas vaginalis: A moving object usually the size of 10 μm in length and 7 μm in width.

Yeast cells: Oval or round in shape having a size of usually 3-5µm and some with budding

Pus cells: Dead white blood cells form pus and the pus cells having a size of usually 12 to 14 µm with lobes.

Clue cells: Clue cells are epithelial cells of the vagina that get their distinctive stippled appearance by being covered with bacteria.

Uses of wet mount or vaginal smear

It is very useful in vaginal infections to find out –

Bacteria
Yeast cells
Trichomonas
Pus cells and
A clue cell is an indicator of bacterial vaginosis.
vaginal pH determination-The normal vaginal pH is 3.8 to 4.5. Bacterial vaginosis, trichomoniasis, and atrophic vaginitis often cause a vaginal pH higher than 4.5.
Whiff test. Several drops of a potassium hydroxide (KOH) solution are added to a sample of the vaginal discharge. A strong fishy odor from the mix means bacterial vaginosis is present.
KOH mount: A sample of the vaginal discharge is placed on a slide and mixed with a solution of potassium hydroxide (KOH). The KOH kills bacteria and cells from the vagina, leaving only yeast for a yeast infection.
It may also be used in a rape investigation to detect the presence of sperm.

Limitation of wet mount or vaginal smear

Infections like chlamydia, genital warts, syphilis, herpes simplex, and gonorrhea can also affect the vagina, but the causative agents of these diseases can not be covered by this wet mount.
Due to the lack of stain, it is difficult to get morphological details.
Inappropriate preparation of the smear may hide parasites.
Improper adjustment of the microscope in relation to the objective may create problems.

Keynotes

A properly prepared wet mount should be thin enough to allow newsprint to be visible.
There is little difference between normal and physiological saline. Physiological saline is 0.85% NaCl were as normal saline is 0.9% NaCl.
Oil immersion examination is also preferred in parasitology for the permanent stained smear of parasites.
Intestinal protozoa can not conform on the basis of a wet mount alone and thus permanent stained smears require to confirm the specific identification of suspected organisms.

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