How long do Ziehl-Neelsen stained samples keep?

How long do Ziehl-Neelsen stained samples keep?

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I have acquired TB slides from my city's health center that are supposedly acid-fast bacteria (AFB) positive. However, when I took a look at it, I see basically no carbol fuchsin stains.

They were individually neatly folded in paper. When I opened them, the stains were slightly wet with immersion oil, facing down. So far, so good (page 6). I've been later informed that these samples were prepared Q1 2019. Based on my research, the staining solutions themselves last up to 12 months (page 33). I can't find any data on how long a stained sample would keep though.

Do they decolorize over time? I expected the AFB to be bright, or at least slightly pink, but I see no such thing. I do see bacilli, though all of them are nearly transparent/methylene blue in color.

Here are some sample images. The first 2 are how I expected AFB to look like, while the next 2 are what I captured (and I think should contain AFB). The last pic is a 10um/div calibration slide for reference.

This TB study might help you: external page

Briefly, they used 6 to 19 months old samples of mostly bad quality (due to their age). The last sentence of the quote below sounds promising though.

The 12,543 sputum smears stored at the national TB reference laboratory for 6 to 19 months were stored at room temperature in a wooden slide box without a cover slip. Most of them had lost their stain reflecting the counterstaining with methylene blue. Restaining of 436 doubtful slides by ZN gave disappointing results throughout [… ]

After a 6 to 19 months storage of ZN stained sputum smears in a non-air conditioned atmosphere, the bright red color of eggs due to carbolfuchsin had virtually disappeared. [… ] In such case, the ZN restaining of sputum smears allows to rediscover AFB that were discolored.

When I did this the last time, we used fresh and few months old samples which looked similar to the first of your images. Just have a look at the paper, I guess.

Drawing a circle on the underside of the slide using a glassware-marking pen may be helpful to clearly designate the area in which you will prepare the smear. You may also label the slide with the initials of the name of the organism on the edge of the slide. Care should be taken that the label should not be in contact with the staining reagents.

  • Bacterial suspensions in broth: With a sterile cooled loop, place a loopful of the broth culture on the slide. Spread by means of circular motion of the inoculating loop to about one centimeter in diameter. Excessive spreading may result in disruption of cellular arrangement. A satisfactory smear will allow examination of the typical cellular arrangement and isolated cells.
  • Bacterial plate cultures: With a sterile cooled loop, place a drop of sterile water or saline solution on the slide. Sterilize and cool the loop again and pick up a very small sample of a bacterial colony and gently stir into the drop of water/saline on the slide to create an emulsion.
  • Swab Samples: Roll the swab over the cleaned surface of a glass slide.

Please note: It is very important to prevent preparing thick, dense smears which contain an excess of the bacterial sample. A very thick smear diminishes the amount of light that can pass through, thus making it difficult to visualize the morphology of single cells. Smears typically require only a small amount of bacterial culture. An effective smear appears as a thin whitish layer or film after heat-fixing.

How long do Ziehl-Neelsen stained samples keep? - Biology

Parasitol Latinoam 61: 117 - 120, 2006 FLAP

Detection of Cryptosporidium oocysts by auramine and Ziehl Neelsen staining methods


* Departamento de Ciências Biológicas e da Saúde, Faculdade de Biologia, Universidade do Planalto Catarinense, Lages, Santa Catarina, Brasil.
** Laboratório de Protozoologia, Faculdade de Medicina Veterinária, Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul,

Cryptosporidium spp is a common intestinal pathogen of animals and humans. It may have an important economic impact on farms and cause potentially zoonotic infections. Fecal specimens were collected from 331 domestic animals (81 beef cattle, 50 sheep, 100 pigs and 100 dogs) and checked for the presence of Cryptosporidium oocysts by way of Ziehl Neelsen and auramine staining methods. An overall positivity rate of 7.5% (25/331) was found, with rates of 10% (10/100) among the dogs and 18.5% (15/81) among the beef cattle. The feces of sheep and pigs tested negative. In beef cattle, 15 and 12 positive samples were detected by the auramine and Ziehl Neelsen staining techniques, respectively, with no statistically significant difference between the two methods. In dogs, the same number of positive samples was found by both techniques.

Key words: Cryptosporidium, domestic animals, auramine, Ziehl Neelsen method.

Members of the genus Cryptosporidium are eukaryotic organisms, including obligate and intracellular parasites. Cryptosporidium has a complex life cycle, including both sexual and asexual reproduction, an auto-infectious cycle, and the ability to complete its development within a single host. The transmission form is a robust, environmentally resistant oocyst, excreted in the stool, which can exist for long periods of time in the environment. Because animals, in particular domesticated livestock, are its primary host, human infection is usually zoonotic 1 . Those at greatest risk are immunocompromised adults and children, especially those with AIDS, children in day care, travelers to endemic regions, dairy or cattle farm workers or their families or contacts, household contacts of cases or carriers, and possibly owners of infected dogs or cats or their neighbors 2-4 .

The genus Cryptosporidium includes 13 species that are currently considered valid, distributed among domestic and wild mammals, birds, reptiles, and fish. Other morphologically distinct species have been found in fish, reptiles, birds, and mammals, but have not been named 5 . Five Cryptosporidium species (C. parvum, C. hominis, C. canis, C. meleagridis and C..felis ) are the causative agents of infection in humans 6 .

C. parvum is a zoonotic pathogen composed of genetically distinct but morphologically identical genotypes. Although large-scale studies of Cryptosporidium infection in dogs have been performed in several countries, the isolates were not accurately identified because of the lack of a method for molecular analysis. It is important to identify the isolates harbored in dogs, which come in close contact with humans, in order to control human cryptosporidiosis 7, 8 .

Cats are a source of cryptosporidiosis for humans, regardless of whether they are immunocompromised or not however, only one human case has been described, in which C. felis was identified using the molecular method 9 .

In pigs, the prevalence of Cryptosporidium parvum has been investigated worldwide, ranging from 1 to 33% 10 , and the following rates have been found: Germany (1.4%), Spain (21.9%), Japan (33.2%) Canada (11%), United States (7.1%) 11, 12 . Preweaning diarrhea, caused by a complex of protozoan organisms including Isospora suis and Cryptosporidium spp., remains a major problem in pigs worldwide 13 . Yet, due to the inadequacies of conventional diagnostics, little is known about the prevalence and significance of Cryptosporidium in pigs 14 .

It is one of the main causes of diarrhea in newborn ruminants, in cattle, sheep, deer 15 , and goats 16, 17 . Cryptosporidium infection in livestock may cause important economic impact to farmers because of its high morbidity and sometimes high mortality rates among farm animals 18 , and disease is well documented. Comparatively, there is less information on the occurrence of cryptosporidiosis in sheep and goats, although the infection is common in these animals with prevalence rates that vary from country to country, often causing death in lambs 12,17,19-21 .

Few studies have been conducted in dogs and a prevalence rate of 9.7% for cryptosporidiosis among Korean dogs while they were monitoring sources of environmental contamination 22 and rate of 9.3% among dogs was demonstrated in Osaka, Japan 8 . By using a molecular method, the authors identified all isolates as C. canis. A study conducted in São Paulo, Brazil, revealed a prevalence rate of 6.8% for cryptosporidiosis among street dogs 23 and of 2.41% in Rio de Janeiro 24 .

The present study was performed to investigate the status of cryptosporidiosis in beef cattle, sheep, pigs and dogs in the rural and urban regions of Lages, state of Santa Catarina, southern Brazil, using two diagnostic methods.

Among 331 fecal samples, 81 were collected from extensively managed beef cattle, 50 from free-grazing sheep, 100 from intensively farmed pigs, and 100 from owned dogs in Lages, state of Santa Catarina, Brazil. Among the beef cattle, 32 were younger than 12 months (17 females and 15 males) and 49 females were up to12 months. Of the dogs, 40 were female and 60 were male, either young or adults, and all of them lived on the outskirts of the urban area. Among the pigs, there were 80 sows and 12 gilts, one male older than 12 months and 7 males aged up to 12 months. Among the sheep, there were 49 ewes and one ram, all of them older than 12 months.

The samples were collected from the rectum of each animal using disposable latex gloves. The samples were submitted to sedimentation of oocysts by centrifugation, in which the sediment was fixed with methanol for 5 minutes and then used for the smearing procedure. The specimens were smeared onto glass slides and stained using the modified Ziehl Neelsen and auramine techniques 25,26 .

The samples stained by the Ziehl Neelsen technique were examined under light microscopy (1,000 X). The auramine-stained smears were analyzed by fluorescence microscopy, after a previous screening (100 X) and later confirmation (400 X).

Fisher's exact test (Graphpad Software, v2.04) was used for the comparison between the two diagnostic methods. An alpha error probability of less than 5% (p < 0.05) was considered statistically significant.


The overall positivity rate was 7.5% among 331 fecal samples examined for Cryptosporidium sp. The comparison between both techniques demonstrated that 25 (7.5%) and 22 (5.7%) samples were positive for the auramine and Ziehl-Neelsen methods, respectively.

Of 100 fecal samples collected from dogs, 10 (10%) were positive, five in female and five in male animals. When these animals were assessed according to age, Cryptosporidium sp. was found in 4 (40%) dogs younger than 12 months and in 6 (60%) adult dogs. The 10% infection rate observed is in agreement with others authors 8,23 .

Among the 15 fecal samples of cattle that tested positive for cryptosporidiosis, 7 (46.6%) belonged to oxen and 8 (53.3%) to cows. In terms of age, positive results were observed in 12 (80%) cattle younger than 12 months and in 3 (20%) cattle older than 12 months.

Auramine detected 25 (100%) positive samples, whereas the Ziehl-Neelsen method detected 22 (80%) positive samples, with no statistically significant difference.

The fecal samples collected from sheep and pigs were negative for Cryptosporidium oocysts.

Several methods, including both flotation and sedimentation, are used for the detection of Cryptosporidium oocysts however, neither of the methods shows any difference 2 . Auramine has a greater affinity for the Cryptosporidium oocyst wall than fuchsin, a red dye used in Ziehl Neelsen staining technique 25 . Auramine-stained oocysts withstand discoloration for 5 minutes, but oocysts stained by the Ziehl Neelsen technique exhibit complete discoloration within the same time frame. Auramine staining has more advantages over the Ziehl Neelsen method, i.e., it is quicker to perform and read, and ideal for population-based studies. Stained slides, if protected from light, can last for months, and can be later stained by the Ziehl Neelsen technique.

The results of the age distribution in this study possibly reflected a bias due to the deviated population structure toward aged animals in rural and urban areas of our city. Direct contact with infected animals is suggested to be an important mode of transmission of Cryptosporidium, which is possibly present in every domestic beef cattle herd in the world with asymptomatic infections and prolonged oocyst excretion by cattle recognized as a major and continuous source of environmental contamination 27,3 .

At least 13 Cryptosporidium species are currently recognized this is based on genotyping and on a limited number of transmission experiments. C. parvum has recently been known to have several different genotypes such as genotype 1, found exclusively in humans and a few other primates, and genotype 2, found in most mammals, including humans 27 although C. hominis, found exclusively in humans, has been well described 5,28 .

The present survey demonstrated that Cryptosporidium infection of calves is important and that further studies are needed to show its relative importance, mainly in the neonatal diarrhea syndrome.

We did not evaluate the genotype of C. parvum in the animals of this study. Future studies will be necessary to verify the infection status of these animals. The positive rate in beef cattle and dogs suggested that these animals could be a source of human infection. Because C. parvum is a major waterborne protozoan pathogen, water contamination should be investigated to protect public health from the risk of transmission of the pathogen.

In addition, these results also highlight the importance of investigating the possibility that other animals also act as reservoir hosts for Cryptosporidium.

1.- CACCIO S M. New methods for the diagnosis of Cryptosporidium and Giardia. Parasitology 2004 46: 151-5. [ Links ]

2.- DUBEY J P, SPEER C A, FAYER R. Cryptosporidiosis of Man and Animals. CRC Press, 1990, 199 p. [ Links ]

3.- YU J R, LEE J K, SEO M, et al. Prevalence of cryptosporidiosis among the villagers and domestic animals in several rural areas of Korea. Korean J Parasitol 2004 42: 1-6. [ Links ]

4.- NYDAM D V, LINDERGARD G, SANTUCCI F, et al. Risk of infection with Cryptosporidium parvum and Cryptosporidium hominis in dairy cattle in the New York City watershed. Amer J Vet Res 2005 66: 413-7. [ Links ]

5.- XIÃO L, FAYER R, RYAN U, UPTON S. Cryptosporidium Taxonomy: Recent Advances and Implications for Public Health 2004 17: 72-97. [ Links ]

6.- GOMEZ-COUSO H, MÉNDEZ-HERMIDA F, ARES-MAZÁS E. Levels of detection of Cryptosporidium oocysts in mussels (Mytilus galloprovincialis) y IFA and PCR methods. Vet Parasitol 2006 140: 44-53. [ Links ]

7.- TAN J S. Human zoonotic infections transmitted by dogs and cats. Arch Int Med 1997 157: 1933-43. [ Links ]

8.- ABE N, SAWANO Y, YAMADA K, et al. Cryptosporidium infection in dogs in Osaka, Japan. Vet Parasitol 2000 108: 185-93. [ Links ]

9.- FAYER R, SANTIN M, TROUT J M, DU EY J P. Detection of Cryptosporidium felis and Giardia duodenalis assemblage F in a cat colony. Vet Parasitol 2006 140: 44-53. [ Links ]

10.- MADDOX - HYTTEL C, LANGKJAER R, ENEMARK H L, VIGRE H. Cryptosporidium and Giardia in different age groups of danish cattle and pigs. Occurrence and management associated risk factors. Vet Parasitol 2006 141: 48-59. [ Links ]

11.- QUILEZ J, SANCHEZ-ACEDO C, CLAVEL A, et al. Prevalence of Cryptosporidium infections in pigs in Aragãn (northeastern Spain). Vet Parasitol 1996 67: 83-8. [ Links ]

12.- OLSON M E,THORLAKSON C L, DESELLIERS L, et al. Giardia and Cryptosporidium in Canadian farm animals. Vet Parasitol 1997 68: 375-81. [ Links ]

13.- DRIESEN S J, CARTLAND P G, FAHY V A. Studies on pre-weaning piglet diarrhoea. Aust Vet J 1993 70: 259-62. [ Links ]

14.- RYAN U M, SAMARASINGHE B, READ C, et al. Identification of a novel Cryptosporidium genotype in pigs. App Env Microbiol 2003 69: 3970-4. [ Links ]

15.- FAYER R, TROUT J M, GRACZYK T K, LEWIS E J. Prevalence of Cryptosporidium, Giardia and Eimeria infections in post-weaned and adult cattle on three Maryland farms. Vet Parasitol 2000 93: 103-12. [ Links ]

16.- KOUDELA B, JIRI V. Experimental cryptosporidiosis in kids. Vet Parasitol 1997 71: 273-81. [ Links ]

17.- MAJEWSKA A C, WERNER A, SULINA P, LUTY T. Prevalence of Cryptosporidium in sheep and goats bred on five farms in west-central region of Poland. Vet Parasitol 2000 89: 269-75. [ Links ]

18.- CASEMORE D P, WRIGHT S E, COOP R L. Cryptosporidiosis - human and animal epidemiology. In: FAYER, R. (Ed.), Cryptosporidium and Cryp-tosporidiosis. CRC Press, Boca Raton, FL, 1997 pp. 65-92. [ Links ]

19.- VIEIRA L S, SILVA M B, TOLENTINO A C, et al. Outbreak of cryptosporidiosis in dairy goats in Brazil. Vet Rec 1997 140: 427-8. [ Links ]

20.- CAUSAPE A C, QUILEZ J, SÁNCHEZ-ACEDO C, et al. Prevalence and analysis of potential risk factors for Cryptosporidium parvum infection in lambs in Zaragoza (northeastern Spain). Vet Parasitol 2002 104: 287-98. [ Links ]

21.- CHALMERS R M, ELWIN K, REILLY W J, et al. Cryptosporidium in farmed animals: the detection of a novel isolate in sheep. Int J Parasitol 2002 32: 21-6. [ Links ]

22.- KIM J T, WEE S H, LEE C G. Detection of Cryptosporidium oocysts in canine fecal samples by Immunofluorescence assay. Korean J Parasitol 1998 36: 147-9. [ Links ]

23.- OLIVEIRA-SEQUEIRA T C G, AMARANTE A F T, FEREIRA T D, NUNES L C. Prevalence of intestinal parasites in dogs from São Paulo State, Brazil. Vet Parasitol 2002 103: 19-27. [ Links ]

24.- HUBER F, BOMFIM T C, GOMES R S. Comparison between natural infection by Cryptosporidium sp., Giardia sp. in dogs in two living situations in the west zone of the municipality of Rio de Janeiro. Vet Parasitol 2005 130: 69-72. [ Links ]

25.- TABOADA J L, CREGO A M, CASAL J F, CRUZ A L. Cryptosporidium, Un Protozoo Asociado Al Sida. Santiago de Compostela, 1993, 126 p. [ Links ]

26.- QUADROS R M. de. Ocorrência de Cryptosporidium (TYZZER, 1907) detectada pelo método de imunofluorescência através da técnica de coloração de auramina em bovinos de propriedades rurais do município de Lages (SC), Brasil. Dissertação de mestrado, 2002. FAVET/UFRGS, Porto Alegre, RS, 53 p. [ Links ]

27.- TZIPORI S, WARD H. Cryptosporidiosis: biology, pathogenesis and disease. Microb Inf 2002 4: 1047-58. [ Links ]

28.- NEVES D P, MELO A L, LUNARDI P M, VITOR R W A. Parasitologia Humana, 11. ed., Atheneu, São Paulo 2005 19: 173-9. [ Links ]

Corresponding author:

Sandra M.T. Marques. Email: [email protected]
Faculdade de Medicina Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 9090, CEP: 91540-000 Porto Alegre, RS, Brasil. Fax: +55 51 33167305.

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A Highly Efficient Ziehl-Neelsen Stain: Identifying De Novo Intracellular Mycobacterium tuberculosis and Improving Detection of Extracellular M. tuberculosis in Cerebrospinal Fluid

Fig 1 Comparison of the conventional and modified Ziehl-Neelsen (ZN) stain for CSF samples from tuberculous meningitis patients. (A and B) The conventional stain shows damaged cellular structure (A) and cell aggregations (B). No intracellular acid-fast bacilli (AFB) are observed with the conventional method. (C) The modified method can concentrate AFB in the CSF. Intracellular AFB are frequently observed in neutrophils (D), monocytes (E), and lymphocytes (F). Arrows show acid-fast-dye-positive AFB. Insets show higher magnification views of AFB or cells indicated by arrows. Scale bars, 20 (A to F) and 5 μm (insets). Fig 2 Intracellular distribution of AFB in neutrophils and monocytes on the modified Ziehl-Neelsen stain. Double labeling of AO (A and E, green) with CD11b (B, red) and ED1 (F, red) shows the intracellular location of AFB in neutrophils (A to D) and monocytes (E to H). AO, CD11b, and ED1 label AFB, neutrophils, and monocytes, respectively. The nuclei are stained by Hoechst 33342 (blue). Panels D′, D″, H′, and H″ show higher magnification views of panels D and H in z axis projections. Scale bars, 20 (A to H) and 5 μm (D′, D″, H′, and H″). Fig 3 Intracellular distribution of AFB in lymphocytes on the modified Ziehl-Neelsen stain. Double labeling of AO (A and D, green) with CD3 (B, red) and CD20 (E, red) shows the intracellular location of AFB in lymphocytes (C and F). The nuclei are stained by Hoechst 33342 (blue). Scale bar, 5 μm.
ABF group (n) % Positive rate (no. of positive samples) of each technique
ZN stainModified ZN stain
Extracellular (48)16.7 (8)100 (48)
Intracellular (48)0 (0)93.8 (45)
Fig 4 Comparison of AFB-positive fields by the conventional (CZN) and modified (MZN) Ziehl-Neelsen stain. Three hundred fields on each slide from 48 CSF specimens were observed. Compared to the ZN stain, which reveals no intracellular AFB-positive fields, the modified ZN stain definitely identifies AFB within the immune cells. Moreover, the modified stain reveals more extracellular AFB-positive fields than the conventional ZN stain.

Acid-Fast Stains

Acid-fast staining is another commonly used, differential staining technique that can be an important diagnostic tool. An acid-fast stain is able to differentiate two types of gram-positive cells: those that have waxy mycolic acids in their cell walls, and those that do not. Two different methods for acid-fast staining are the Ziehl-Neelsen technique and the Kinyoun technique. Both use carbolfuchsin as the primary stain. The waxy, acid-fast cells retain the carbolfuchsin even after a decolorizing agent (an acid-alcohol solution) is applied. A secondary counterstain, methylene blue, is then applied, which renders non–acid-fast cells blue.

The fundamental difference between the two carbolfuchsin-based methods is whether heat is used during the primary staining process. The Ziehl-Neelsen method uses heat to infuse the carbolfuchsin into the acid-fast cells, whereas the Kinyoun method does not use heat. Both techniques are important diagnostic tools because a number of specific diseases are caused by acid-fast bacteria (AFB). If AFB are present in a tissue sample, their red or pink color can be seen clearly against the blue background of the surrounding tissue cells (Figure 5).

Using Microscopy to Diagnose Tuberculosis

Figure 5. Ziehl-Neelsen staining has rendered these Mycobacterium tuberculosis cells red and the surrounding growth indicator medium blue. (credit: modification of work by American Society for Microbiology)

Mycobacterium tuberculosis, the bacterium that causes tuberculosis, can be detected in specimens based on the presence of acid-fast bacilli. Often, a smear is prepared from a sample of the patient’s sputum and then stained using the Ziehl-Neelsen technique (Figure 5). If acid-fast bacteria are confirmed, they are generally cultured to make a positive identification. Variations of this approach can be used as a first step in determining whether M. tuberculosis or other acid-fast bacteria are present, though samples from elsewhere in the body (such as urine) may contain other Mycobacterium species.

An alternative approach for determining the presence of M. tuberculosis is immunofluorescence. In this technique, fluorochrome-labeled antibodies bind to M. tuberculosis, if present. Antibody-specific fluorescent dyes can be used to view the mycobacteria with a fluorescence microscope.

Think about It


Figure 3. The use of protein-based blocking agents reduces nonspecific staining.

If you are going to use antibodies to label structures in your fixed and permeabilized cells, using a blocking solution before your primary antibody step can be helpful. Blocking is usually performed with a solution containing an excess of protein that serves to reduce the amount of nonspecific binding in your sample. This can be important if your primary or secondary antibody has a tendency to interact with molecules in the sample that are not your target. Reduction of nonspecific “background” staining, most likely due to hydrophobic interactions between the antibody and non-target molecules, will make it easier for you to identify a positive signal and will give you a cleaner end result.

The most common types of blocking solutions for ICC are 3% (w/v) bovine serum albumin in PBS and/or a 10% (v/v) solution of heat-inactivated species-specific serum in PBS, where the serum species matches the species of the secondary antibody. For example, if you are using a mouse anti–rabbit IgG secondary antibody, chose normal heat-inactivated mouse serum to make your blocking reagent.

Figure 4. Protein-based blocking agents help reduce non-specific staining. Antibodies are able to displace the blocking proteins to form a high-affinity bond with their epitopes, while blocking proteins prevent low-affinity antibody interactions elsewhere in the sample.

You’ll want to incubate your sample in blocking solution for at least 60 minutes, but it can also be left overnight at room temperature or in the fridge. Once you’re done with the blocking step, if you’re not going to label with antibodies, it’s important to remove the excess blocking solution by washing with PBS.

It can be difficult to figure out where something went wrong in a multi-step process, and immunofluorescence is no exception. The most likely causes of a poor result in immunofluorescence are the primary antibody (either type and/or concentration) or secondary antibody (concentration). See antibody labeling for suggestions on experimental controls to help you optimize your results.

Microscopy Solutions for Histology and Histopathology

Pathology, histopathology or histology aims to study the manifestation of disease by microscopic examination of tissue morphology. In pathology, the sample to be examined under the microscope usually is the result of a surgery, biopsy or autopsy after fixation, clearing/embedding and sectioning of the tissue specimen. Alternatively, frozen section processing with a cryostat is done when rapid results are required (e.g. during surgery) or fixation would be detrimental to target structures such as lipids or certain antigens. The tissue sections after fixation and wax embedding are typically cut into two to five micron thin slices with a microtome before staining and transfer to a glass slide for examination with a light microscope. Typical specimens in pathology are colon, kidney, pancreas, cervix, lung, breast, prostate, or connective tissue.

While various staining procedures for human/animal and plant tissues have been developed as early as the 17th century it was the German physician Rudolf Virchow who is being considered the father of modern histopathology. Virchow realized the potential of the emerging new microscope techniques of the 19th century for his groundbreaking research, published a vast amount of scientific writing and created an impressive collection of thousands of histopathological sample slides, thus building the foundation of modern histology and cancer research.

Histology slide preparation begins with fixation of the tissue specimen. This is a crucial step in tissue preparation, and its purpose is to prevent tissue autolysis and putrefaction. For best results, the biological tissue samples should be transferred into fixative immediately after collection, usually in 10% neutral buffered formalin for 24 to 48 hours. After fixation, specimens are trimmed using a scalpel to enable them to fit into an appropriately labelled tissue cassette that is stored in formalin until processing begins.

The first step of processing is dehydration, which involves immersing your specimen in increasing concentrations of alcohol to remove the water and formalin from the tissue. Clearing is the next step, in which an organic solvent such as xylene is used to remove the alcohol and allow infiltration with paraffin wax. Embedding is the final step, where specimens are infiltrated with the embedding agent – usually paraffin wax which provides a support matrix that allows for very thin sectioning. A microtome is used to slice extremely thin tissue sections off the block in the form of a ribbon, following histochemical staining (typically haematoxylin and eosin - “HE stain”) to provide contrast to tissue sections, making tissue structures better visible and easier to evaluate. In certain cases immunohistochemical stainings (IHC), such as HER2 or Ki-67, are required for further analysis.

IHC staining protocol for whole mount samples

Whole mount staining is the staining of small pieces of tissue – usually embryos – without sectioning.

Whole mount staining is very similar to immunocytochemistry (ICC) or staining of cryosections. If an antibody has been used successfully on cryosections (this does not include paraffin-embedded sections), then the antibody should work for a whole mount embryo.

The difference is that the sample being stained is much larger and thicker than a normal section on a slide. Therefore, incubations for fixative, blocking buffer, antibody, wash buffer, permeabilization and substrate color development will need to be much longer to allow for permeabilization right into the center of the sample.

The timing of these steps will need to be optimized for your experiments, but the details in these protocols provide a guideline.


Whatever fixative you have successfully used in IHC-Fr with your antibody should also be suitable for whole mount. However, most researchers use 4% paraformaldehyde (PFA).

Although this concentration of PFA is very low, this has to be left on for a long period of time on whole mount samples to allow permeabilization to the center of the sample. Therefore, this will not be suitable for all antibodies, as the protein cross-linking formed by the fixative may block access of the antibody to the epitope.

Normally, in IHC-P you could perform antigen retrieval. This is not possible on embryo samples as the heating procedure would destroy the sample. If PFA fixation does not work for the whole mount tissue, then there is a possibility the antibody is sensitive to the protein cross-linking, and you will require another fixative. Methanol is a popular second choice of fixative when optimizing whole mount procedures.

Zebrafish embryo fixation and preparation requires extra steps to ensure the egg membrane is permeabilized.

Obtaining images

Some researchers view and obtain images of embryos as they are. The whole embryo can be imaged while floating in glycerol buffer in a petri dish, before mounting. If small enough, the whole embryo can be mounted in glycerol before setting in a cover slip. In this case, grease should be used around the corner of the cover slip to help keep it in place .

Embryos can also be set in gelatin and sectioned if it is difficult to obtain a clear view of the staining through the whole embryo (particularly at larger late embryo stages or larger tissue samples).

If immunofluoresent labeling is used, then confocal microscopy can be a useful tool to scan through the embryo, rather than sectioning the whole embryo onto separate slides after staining.

Choosing the age of the embryo

As the embryo grows, it will become too large to stain. The various reagents, including fixative, antibody and developing solution will not be able to permeate to the center of the sample, and the number of stained cells will make obtaining a clear image very difficult. However, larger and older embryos can be dissected into segments before staining if necessary.


M. leprae replicates intracellularly, typically within skin histiocytes, endothelial cells, and the schwann cells of nerves. Cell mediated immunity (CMI) plays major part in determining the response of the host to leprosy.

  1. Tuberculoid leprosy: very few acid-fast bacilli in skin smear (paucibacillary disease): Cell-mediated immune (CMI) response is adequate and lepromin test is positive.
  2. Lepromatous leprosy: large numbers of Mycobacterium leprae chiefly in masses within the lepra cells, often grouped together like bundles of cigars or arranged in a palisade (multibacillary disease).The cell-mediated immune (CMI) response to organism is poor and the lepromin test is negative.

Ridley and Jopling (1966) have introduced a scale for classifying the spectrum of leprosy into five groups:

  1. Tuberculoid (TT)
  2. Borderline Tuberculoid (BT)
  3. Borderline (BB)
  4. Borderline Lepromatous (BL)
  5. Lepromatous (LL)

According to World Health Organization (WHO), leprosy is divided into two groups, paucibacillary and multibacillary.

Comparison of tuberculoid and lepromatous leprosy

Lepromin Skin Test:

The lepromin skin test is not used to diagnose leprosy but to determine what type of leprosy a person has. Lepromin skin test is similar to tuberculin test. An extract of M.leprae is injected intradermally and induration is observed 48 hours later in those whom a cell-mediated immune response against organism exists.

Lepromin test is employed mostly for the following two purposes.

1. To classify the lesions of leprosy patients.

2. To assess the prognosis and response to treatment.

Microbiology Interview Questions And Answers

  • Simple stain: where only one stain is used and all bacteria are stained similarly. Eg: F1ethylene blue, dilute carbol fuchsin
  • Differential staining: where different bacteria stain differently to a common staining technique depending on their physiological properties. Eg: Gram&rsquos stain and Acid fast staining
  • Special stain: where structures of bacteria like spores. granules. capsule etc are demonstrated. Eg: silver impregnation technique for demonstration of spirochetes. Feulgen stain for demonstration of nucleus. Sudan black stain for demonstration of lipid vacuoles. Ryu&rsquos stain for demonstration of flagella. Albert&rsquos stain for demonstration of metachromatic granules.
  • Negative staining: where the background is stained with an acidic dye such as India ink or Nigrosin. Used for demonstration of capsules.

Stains are classified based on the pH of their chromophore (color bearing ion) into acidic, basic and neutral. Acidic dyes have anionic chromophore

eg.. sodium+ eosinate-. Basic dyes have cationic chromophore eg.. metFiylene blue+ chloride-. Acidic dyes combine more strongly with cytoplasmic components of bacteria, especially the nucleus that is basic in nature. Neutral dyes have both acidic and basic component that nullity each other.

They are Romanowsky&rsquos stain and are used in staining parasitic forms. Stains can be either natural (eg: carmine and hematoxylin) or coal-tar derivatives /aniline stains (eg: methylene blue. crystal violet). Supravital (cells removed from the body) and intravital (cells still a part of the body).

LoetTler&rsquos methylene blue solution treated with Potassium hydroxide turns into Polychrome methylene blue after prolonged storage with shaking. Used in McFadyean&rsquos reaction for Bacillus anthracis in blood films and demonstration of metachromatic granules of Corynebacterium diphtheriae.

Hans Christian Gram invented this stain in 1884. The original formulation was Aniline Gentian violet. Lugol&rsquos iodine, absolute alcohol and Bismark brown.

Cell wall theory: Cell wall of Gram positive bacteria are 40 times thicker than those of Gram negative cells, hence they are thought to help retain the dye-iodine complex.
Lipid Content Theory: Cell envelope of Gram negative bacteria contains an additional membrane (outer membrane). hence containing more lipids than Gram positive bacteria. Acetone or alcohol dissolves the lipid thus forming large pores in Gram negative bacteria through which the dye-iodine complex leaks out. Alcohol/acetone dehydrates Gram positive bacteria shrinking the cell wall and the closing the pores.
Magnesium Ribonucleate Theory: A compound of magnesium ribonucleate and basic protein concentrated at the cell membrane helps Gram positive bacteria retain the primary dye. Gram negative bacteria do not possess this substance.
Cytoplasmic pH Theory: The cytoplasm of Gram positive bacteria are said to be more acidic (2) than those of Gram negative ones (3). Hence the dye is said to bind with more affinity to Gram positive cells.

It is the cytoplasm (especially the nucleic acid) that gets stained and not the cell wall. Presence of an intact cell wall is important for retaining Gram positivity. Cell wall deficient forms such as Mycoplasma and L forms are Gram negative.

  • Extremely slender bacteria such as Treponema
  • Cells containing waxy substances impermeable to stain such as Mycobacteria
  • Minute intracellular bacteria such as Chlamydia and Rickettsia
  • Cell organelles such as capsule. spore. flagella etc
  • Primary stain: Crystal violet. Methyl violet and Gentian violet
  • Mordant: Grams iodine, rarely Lugols iodine
  • Decolorizer: Alcohol, acetone. acteone-alcohol mixture (1:1)
  • Counterstain: Dilute carbol fuchsin. safranin, neutral red. (Sandiford stain ror Gonococcj
  • When over-decolourized by either prolonged exposure to decolourizer or using acetone alone.
  • When cell wall gets damaged by exposure to lysozyme or cell wall acting antibiotics such as Penicillin.
  • Old cultures, where cell wall is weakened or action of autolytic enzymes
  • Those bacteria that are phagocytosed. where cell wall is acted upon by lysosomal contents

Decolourization is the most important step as this step differentiates between Gram positive and Gram negative bacteria. Over-decolourization can result in Gram positive bacteria appearing Gram negative and under-decolourization can result in Gram negative bacteria appearing Gram positive.

  • Rapid presumptive diagnosis of diseases such as bacterial meningitis
  • Selection of empirical antibiotics based on Gram stain finding
  • Selection of suitable culture media based on Gram stain finding
  • Screening of quality of clinical specimens. such as sputum that should contain many pus cells and few epithelial cells
  • Counting of bacteria
  • Appreciation of morphology and types of bacteria in a clinical specimen
  • Kopeloll&rsquo and Beerman&rsquos (Primary stain: Methyl violet. decolourizer: acetone or alcohol-acetone mixture 1:1)
  • Jensen&rsquos (Primary stain: Methyl violet, decolourizer: absolute alcohol. counterstain: Neutral red)
  • Preston and Morrell&rsquos (Primary stain: crystal violet. decolourizer: iodine-acetone)
  • A.igert&rsquos (Primary stain: Carbol gentian violet. decolourizer: Aniline-xylol). This is used to stain tissue sections.

Certain bacteria or their structures have the ability to retain the primary dye (strong carbol fuchsin) and resist clecolourization by weak mineral acids such as H2S04. HCI. Such bacteria or their structure are termed acid fast and this property is termed acid fastness. There are two types of acid fast staining, the hot method and the cold method. The hot method (Ziehl-Neelsen) involves heating the slide while the cold methods such as Kinyoun&rsquos and Gabbett&rsquos do not involve heating the slide.

Ehrlich in 1882 discovered acid fastness. The original method involved staining with aniline-gentian violet and decolourization with strong nitric acid.

It was later improved by Ziehl and Neelsen.

The cell walls of Mycobacterla are made up of waxy substance, Mycolic acid that Is relatively Impermeable to ordinary stainIng techniques. But, by apphcation of heat and a mordant (phenol), the cell can be stained The purpose of heating is to soften the waxy material of the cell wall and allow the stain to enter the cell. Basic fuchsin is more soluble in phenol and phenol is a better solvent for lipids and waxes.

  • Primary stain: Strong Carbol Fuchsin (contain Basic fuchsin and Phenol)
  • Decolourizer 20% sulphuric acid
  • Counterstain LoelTler&rsquos Methylene blue or 1% Malachite green, Picric acid for color-blind workers

3% HCI in 95% alcohol (methylated spirit). This is useful in dilrerentiating saprophytic Mycobacteria from pathogenic Mycobacteria Pathogenic Mycobacteria are both acid and alcohol fast but saprophytic Mycobacterla are only acid-fast Saprophytic Mycobacterla can get declourized by alcohol. 95% alcohol can be used as a secondary decolorizer after decolourizing with acid Especially used in staining smears prepared from urine that may contain Mycobacterium smegmatis.

  • Mycobacterlum leprae - 5% H2S04
  • Oocysts of Cryptosporidium. Isospora - 1 % H2S04
  • Tissue sections containing Actinomyctes. Nocardia - 1 % H2S04
  • Cultures of Nocardia - 0.5% H2S04
  • Bacterial spores - 0.25-0.5% H2S04

The two methods namely Kinyoun&rsquos and Gabbetts dont involve heating of slides, hence called cold methods. Heating is substituted by increased concentration of phenol and prolonging the duration of staining. Kinyoun&rsquos method is favoured for detection of Cryptosporidium oocysts in fecal samples. Gabbetts method has decolourizer and counterstain in one solution.

For uniform distribution of heat, or else the slide may break.

  • A new slide must be used for every specimen. because scratch marks may give false positive.
  • A uniform smear from thick portion of the sputum must be made.
  • Staining jars should not be used to staining smear as there is risk to cross contamination
  • Fresh blotting paper must be used for each smear for drying the slide to prevent transfer from one slide to another.

At least 100 oIl Immersion fields must be viewed before declaring the smear as negative The sensitivity of smear Is low because It requires the presence of 104 bacillilml to be smear positive. If the number of bacilli is less than this, the chances of detecting them are less In such a case, the sample should be subjected to concentration techniques such as Petroff s method If the smear Es positive for AFB. it should be counted/graded Failure to detect any AFB does not rule tuberculosis Grading of smears has prognostic value.

Smears are graded depending on the number of bacilli seen

  • 3-9 bacilli/entire smear: +
  • 10 bacilli/entire smear ++
  • 10 bacilli/in most oil Immersion fields: +++

Sputum smears for Mycobacterla can be stained by fluorescent dyes such as Auramine and Rhodamlne as they have affinity for mycolic acid In their cell walIs The fluorescent microscopy is useful in screening large number of specimens. Large area of smear can be quickly observed that too under high power dry objective.

Beaded appearance is used to describe the appearance of Mycobacterla when the cell doesn&rsquot stain uniformly. showing stained and unstained regions. These forms are common in Mycobacterium tuberculosis while Mycobacterium bovis stains uniformly. Most saprophytic Mycobacteria stain uniformly.

Metachromatic granules are pol&rsquoymetaphosphate reserves produced by Corynebacterium diphtheriae in nutritious medium. These granules are also known as Babes Ernst granules. &lsquoblutin granules. Polar bodies etc. They are called metachromatic granules because of they exhibit metachromasia.

a property where the granules appear in a colour different from that of the dye used When stained with polychrome methylene blue, they appear purple They are produced In abundance In serum containing medium such as Loeffler&rsquos serum slope.

Albert&rsquos stain. Neissers stain, Ponder&rsquos stain and Pugh&rsquos stain They can be demonstrated as retractile bodies in wet mount or slightly more gram positive structures in Gram stain.

The bacdh are arranged at angles to each other resembling English letter V or L or Chinese letter (cuneiform) pattern because the daughter cells doWt separate completely after cell dMslon (binary rission).

Solution A(1) contains Toluidine blue, Malachite green, Glacial acetic acid and Alcohol while solution 8(2) contains iodine and potassium iodide In distilled water.

The process of kdling all hying forms including spores is called sterilization and the process of killing of only the vegetative form of pathogenic bacteria as well as other microbes is disinfection

121°C for 15 minutes at 15 pounds per square inch of pressure

Glassware&rsquos, metallic instruments like scissors and forceps, swabs. powder. oils and grease.

Culture media, gloVes. cotton and clothes.

By High Elliciency Particulate Air (HEPA) filters.

Phenol. Lysol, Formaldehyde. Sodium hypochlorite.

Antiseptics are mild disinfectants that can be safely used on skin and mucous membranes.

Quaternary ammonium compounds are positively charged polyatomic ions, which concentrate at the cell surface and alter the physical and chemical properties of the membrane, thus killing the cefl. Examples inlcude Benzalkonium chloride and Cetrimonium bromide.

The active ingredient of Dettol is chloroxylenol whereas Savlon contains a combination of Cetrimide and Chiorhexidine.

The active ingredients include Chlorhexidine. Triclosan. Thymol. Cetylpyridinium Chloride, and alcohol. The composition varies across brands.

These are the chemicals used for sterilization. They are 2% Gluteraldehyde (cidex). Ethylene Oxide (EO). Formaldehyde + steam and Beta &mdash Propiolactone (BPL).

  • Tincture Iodine - 2% of Iodine in 70% alcohol - lodophore - Povidone Iodine.
  • Name some antiseptics.
  • Chlorhexidine. Chloroxylenol. spirit (70% alcohol), tincture of Iodine. H202.

Sodium hypochlorite or Calcium hypochlorite

By treating them with disinfectant, boiling or autoclaving and finally by incineration

Gamma rays. Electron beams and Ethylene oxide

Glutaraldehyde or a combination of peracetic acid and hydrogen peroxide can be used.

Alkylation (hydrogen atom is replaced with an alkyl group) of protein. DNA. and RNA afFects bacterial metabolism and replication. EQ gas (8.5%) is often mixed with stabilizers such as CO2 (91 5%) or hydrochlorofluorocarbons (HCFC). This requires high humidity (40-80%) and long exposure times (1-6 hrs).

Ducking Is a process of inactivation of Anthrax spores in animal products such as wool, hairs or bristles. It was introduced by Elmhirst Duckering, an enginer at wool factory. This is a live-step process. each lasting for 10 minutes and carried out at 40.5°c.

  • immersion in 0.25-0.3% alkali
  • immersion in soapy water
  • immersion in 2% formaldehyde
  • secondimmersion in 2% formaldehyde
  • rinsinq in water

Porcelain filters. Seitz (asbestos) filters. Sintered glass filters. Membrane filters and HEP filters.

It disinfects and solidifies egg and serum containing media such as U medium and LoelTiers serum slope.

Watch the video: ziehl neelsen Stain Clear u0026 Complete Overview (August 2022).