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What is the Giemsa staining of chromosomes?

What is the Giemsa staining of chromosomes?


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I got the question in my exam and wrote the following and I do not understand what is wrong in it:

Giemsa staining is a staining method to stain particularly malaria and other parasital diseases. G-bands occur because Giemsa stain consists of A,T rich material i.e. poor gene such that dark and white bands occur. Each chromosome has an unique reaction to Giemsa staining so G-bands occur.

0 points. I do not understand what's wrong with it, since in their comments about the same question in my first exam they wrote also the extra questions: What are G-bands? How are they formed and why? This time I answered the given things and got zero mark.

Probably, the mistake was that I did not answer to the question in the scope of medical Biology in some way. However, I am not exactly sure what it is exactly.

How would you answer to the question when you know that the course was about medical biology?

Please, add the tag Giemsa.


Giemsa is not a particular methid to stain malaria or any other parasite. It stains DNA. As such, it can be used to stain any DNA-containing organism, or, in other words, any known cell.

Regarding its particular use in chromosomal banding, you can refer to many online resources, such as this one of the University of Washington:

Chromosomes in metaphase can be identified using certain staining techniques, so called banding

(… )

G-bands are most commonly used. They take their name from the Giemsa dye, but can be produced with other dyes. In G-bands, the dark regions tend to be heterochromatic, late-replicating and AT rich. The bright regions tend to be euchromatic, early-replicating and GC rich.


Probably they want something like from here:

For differentiate nuclear and/or cytoplasmic morphology of platelets, RBCs, WBCs and parasites. In wright- and Giemsa-stain: the cytoplasm appears blue and the nucleus is relatively large, eccentrically located and red. The distinct, rod-shaped, red-staining kinetoplast (a specialized mitochondrial structure) contains extranuclear DNA arranged as catenated minicircles and maxicircles.

In my opinion, the question is not exact if they want the above thing. It should have been: What is the Giemsa staining of chromosomes morphologically? if the above thing correct.

It was the first medical biology course so answer should be from it.


Giemsa Stain: Principle, Procedure, Results

Giemsa stain is a type of Romanowsky stain, named after Gustav Giemsa, a German chemist who created a dye solution. It was primarily designed for the demonstration of malarial parasites in blood smears, but it is also employed in histology for routine examination of blood smears.

Uses of Giemsa Stain

  • Giemsa stain is used to obtain differential white blood cell counts.
  • It is also used to differentiate nuclear and cytoplasmic morphology of the various blood cells like platelets, RBCs, WBCs.
  • In Microbiology, Giemsa stain is used for staining inclusion bodies in Chlamydia trachomatis, Borrelia species, and if Wayson’s stain is not available, to stain Yersinia pestis. Giemsa stain also is used to stain Histoplasma capsulatum, Pneumocystis jiroveci, Klebsiella granulomatis, Talaromyces marneffei (formerly called Penicillium marneffei) and occasionally bacterial capsules.
  • This stain is also used in cytogenetics to stain the chromosomes and identify chromosomal aberrations. It is commonly used for G-banding (Giemsa-Banding)

Principle of Giemsa Stain

Giemsa stain is a differential stain and contains a mixture of azure, methylene blue, and eosin dye. It is specific for the phosphate groups of DNA and attaches itself to where there are high amounts of adenine-thymine bonding.

Azure and eosin are acidic dye that variably stains the basic components of the cells like the cytoplasm, granules etc.

Methylene blue acts as the basic dye, which stains the acidic components, especially the nucleus of the cell.

Methanol act as a fixative as well as the cellular stain. The fixative does not allow any further change in the cells and makes them adhere to the glass slide.

Composition of Giemsa Stain

PROCEDURE

A. For In-house preparation of stain:

  1. Weigh the required amount of powder stain, and transfer to a clean, dry 1litre capacity bottle. Add methanol and mix well.
  2. Measure and add glycerol and mix well.
  3. Place the bottle of stain in water bath at 50-60°C or at 37°C for up to 2hours with frequent mixing.
  4. Label the bottle and store in a cool, dark place with a firm stopper.

NOTE: If water gets in contact during any steps of preparation of stain, the stain gets spoilt, therefore use, dry glassware and store in conditions where there would be no water contact.

  • Filter the stain using Whatman filter paper no.1 and dilute with water buffered to pH 7.2 to make working solutions

B. For staining slides

The method for staining, concentration and timing of stain used varies according to the purpose, for example, thin blood smears use 1:20 dilution of stock whereas for thick blood smear 1:50 dilution is used.

For Thin blood smear

  1. Fix air-dried film in absolute methanol by dipping the film briefly (two dips) in a Coplin jar containing absolute methanol.
  2. Remove and let air dry.
  3. Stain with diluted Giemsa stain (1:20, vol/vol) for 20 min (For a 1:20 dilution, add 2 ml of stock Giemsa to 40 ml of buffered water in a Coplin jar).
  4. Wash by briefly dipping the slide in and out of a Coplin jar of buffered water (one or two dips).
    Note: Excessive washing will decolorize the film.
  5. Let air dry in a vertical position. Observe under the microscope first at 40X and then using oil immersion lens

For Thick blood smears

  1. Allow the film to air dry thoroughly for several hours or overnight. Do not dry films in an incubator or by heat, because this will fix the blood and interfere with the lysing of the RBCs.
    Note: If a rapid diagnosis of malaria is needed, thick films can be made slightly thinner than usual, allowed to dry for 1 hour, and then stained.
  2. DO NOT FIX.
  3. Stain with diluted Giemsa stain (1:50, vol/vol) for 50 min (For a 1:50 dilution, add 1 ml of stock Giemsa to 50 ml of buffered water in a Coplin jar)
  4. Wash by placing the film in buffered water for 3 to 5 min.
  5. Let air dry in a vertical position and observe under the microscope first at 40X and then using oil immersion lens

For Chlamydia trachomatis

Follow the aforementioned steps but with the dilute stain of 1:40 dilution (add 0.5 ml stock Giemsa solution to 19.5 ml buffered water) and leave the stain for 90-120 minutes.


Karyotyping, Giemsa Staining - (Aug/26/2014 )

I am preparing chromosomes for karyotyping, because I need to know the chromosome number of my study organism (ant).

So I just need to count them.

I used a chromosome preparation protocol suitable for ants (including colchicin treatment) and did a standard Giemsa stain.

But I never see clear metaphase chromosomes, they more like round bubbles and they stick really close together, so that I am not able to count. 

Now I read that Giemsa is making chromosomes bulky. Perhaps if my chromosomes are very short, this leads to their round appearance?

I do not think that its a problem with the microscope, but I am not sure, as pictures do not get too clear. (I have 1600x magnification available)

Can somebody recommend another method of staining? Or help me anyway?

Thank you so much in advance!

First of all, it seems to me that your photo is out of focus. Are you experienced in using a microscope? Have you added immersion oil on the slide (your lens is oil-immersed, am I right?)? Nevertheless, I would suggest cleaning the objective lens, the stage and the illuminator very well.

Apart from these, you are right, your metaphase is poorly spread and the chromosomes are not in a great state. I could suggest some stuff but everything is based on human cell cultures (don't know if they apply. ) To get well-spread metaphases I would suggest experimenting with ΚCL incubation (longer incubations). I use pre-warmed KCL and add it to the cell suspension slowly  under gentle vortexing.

Mitogen incubation could also affect chromosome appearance and metaphase number.

Another thing that might affect chromosome appearance (elongated chromosomes) and metaphase spreading are slide spreading conditions (humidity, room temperature, as well as the height from here you drop your cell suspension on the slides). Slides should be cold (stored at -20C in methanol). According to my experience I get  well- spread long chromosomes at about 65% humidity, at room temperature less than 22C. Drying time shouldn't take too long. You can use a blow dryer (at the lowest scale) but not to close because you will destroy the chromosomes (you have to experiment on that). By blowing only at a specific angle you can also improve your metaphase spreading. Slides shouldn't also dry too quickly.

You also have to keep in mind that some drugs and medical conditions affect chromosome structure. But I guess this does not apply to your case.

You could also add thymidine in your cell culture so as to get elongated chromosomes.

I don't know if I helped or confused u even more but metaphase spreading is a try and see thing. Spread one slide at a time, take note of all the conditions, check on the microscope. if it doesn't work make some changes and try again. Good luck! :) 

thanks so much for your help!

Your totally right, I have to work on using the microscope.

Yes, I have added immersion oil on the slide.

I will try to clean everything and improve the pictures.

Thanks for all your suggestions, I will definitely try out some of them. 

The major problem is, that I do not work with cell culture.

For chromosome preparation, I dissect male ants, isolate their testis und pull testis into pieces to release cells (everything is happening on the slide already). 

I incubate in colchicine solution to stop meiosis in metaphase. 

KCl isn´t included in my protocol, but instead a hypotonic solution of Na3Citrat x 2H2O

Do you think the kind of hypotonic solution makes a difference?

Consequently, I do not drop anything on the slide, as testis are so tiny I would definitely lose them in a tube.

Nevertheless, I will try storing slides in -20°C Methanol, as you suggest.

Humidity is a good point as well, it´s also mentioned in the protocol I use, I will try that as well.

I am not sure about 2 things you mentioned: mitogen and thymidin:

I do not use any mitogen, as cells in male testis are dividing anyway. 

Do you think I should add it to the protocol?

As far as I know thymidin is inhibiting DNA synthesis, right?

As I already use colchicine to arrest in metaphase, I do not know if I should try?

After colchicine treatment, I use three different fixatives stepwise: 2 different Ethanol-GAA mixtures and finally GAA purely.

Than I apply a standard Giemsa stain. 

So you think it´s not a problem with the stain but with the chromosome preparation, right?

I am very grateful for your help!

I know that want I´m doing is totally different from human cell culture, but I will try out your suggestions!

Yes,based on my experience Giemsa is not the problem. 

 The type of the hypotonic solution shouldn't hypothetically matter. Just be careful, cause prolonged incubation might also lead to chromosome loss (metaphases might spread too much so will not be able to accurately determine chromosome number/metaphase).  

I am sorry, I meant mitotic inhibitors such as colchicine. I have noticed that prolonged incubation, affects the total number of metaphases and chromosome morphology.

Thymidine and methotrexate as well as other factors are used in order to obtain elongated chromosomes (important in karyotyping analysis by G- or R-banding). But I think that this is the last thing to try. As I can understand u just want to count the number of chromosomes per metaphase so you don't really need to get elongated chromosomes but just well-spread complete metaphases.

Another important thing that I forgot to mention is the time of staining. I always allow my slides to dry for 24 hours and then I stain them. I have noticed that I get "prettier" chromosomes. Sometimes I also dry them at 90 C or 65C for 2-3 minutes just before staining (One slide at a time, so as to find what works best..). 

I have found some papers on the internet that might help u more than my suggestions, although u might propably already have them because they are specific for chromosome preparations from ants.

Thanks again for the help and for the papers!

I partly know the papers, the protocol I am using is actually from the guy of the third paper, so really ant specific.

Yes, my only goal is to count chromosomes, to get to know the chromosome number of the species. 


Ocular allergy

Neal P Barney , . Frank M Graziano , in Ocular Disease , 2010

Pathophysiology

Giemsa stain of scrapings from the upper tarsal conjunctiva will reveal eosinophils. Eosinophils (which are never found in normal tissue) as well as a large number of mononuclear cells are present in the substantia propria in AKC. These eosinophils are found to have increased numbers of activation markers on their surface. 42 Fibroblast number is increased and there is an increased amount of collagen compared to normal tissue. This finding is likely critical to the sight-threatening nature of the disease. The substantia propria also demonstrates an increased ratio of CD4+ to CD8+ T cells, B cells, human leukocyte antigen (HLA)-DR staining, and Langerhans cells. 39 The T-cell receptor on lymphocytes in the substantia propria is predominantly of the α or β subtype. 39 The T-cell population of the substantia propria includes CD4+ memory cells. 43 Th2 cytokines predominate in allergic disease yet lymphocytes with Th1 cytokines have been found in the substantia propria of AKC patients. 44

Laboratory manifestations in AKC are shown in Table 13.2 . The tears of patients with AKC contain increased amounts of IgE antibody, ECP, T cells, activated B cells, eotaxin, eosinophil-derived neurotoxin (EDN), soluble IL-2 receptor, IL-4, IL-5, osteopontin, macrophage inhibitory factor (MIF), and decreased Schirmer's values (56% less than 5 mm). 44–47 A dysfunctional systemic cellular immune response is demonstrated by reduction or abrogation of the cell-mediated response to Candida, and an inability of some patients to become sensitized to dinitrochlorobenzene. 48 Additionally, aberrations of the innate immune response are suggested by increased incidence of colonization with Staphylococcus aureus. Isolation of S. aureus from the eyes (conjunctiva, cilia, lid margin) of 80% of AKC patients (but not from control patients) has been reported. 49 While it is not known to what extent this contributes to the ocular surface inflammation, specific IgE antibodies to S. aureus enterotoxins have been detected in tears from AKC patients. 50 Furthermore, the potential of S. aureus cell wall products to activate the conjunctival epithelium has been suggested in vitro in studies reporting expression and activation of the innate immune receptor, Toll-like receptor-2 via an extract from S. aureus cell wall. 51 Immunostaining demonstrated that conjunctival epithelial cells from AKC patients expressed significantly more Toll-like receptor-2 than nonallergic patients. Despite these in vivo findings, AKC patients who improve with treatment are not found to have change in the rate of colonization with S. aureus bacteria. 52 Serum of AKC patients has been found to contain increased levels of IgE, 29 IgE to staphylococcal B toxin, 52 ECP, 53 EDN, 54 and IL-2 receptor. 55


Chromosome Banding..

We have seen that the chromosome is condensed form of DNA. This condensation requires different histone and non-histone proteins. Each chromosome have reproducible ultrastructure.The study of chromosomes is called cytogenetics.

Fig 1: Structure of a Chromosome

The pictures of stained chromosomes of the cell can be photographed and arranged according to shape, structure and sizes to form a karyogram. When subjected to different treatments before staining, the chromosomes develop different dark and light regions in form of bands. The banding pattern can be used to identify homologous chromosome and detect different types of chromosomal rearrangements abnormalities.

Fig 2: Procedure for chromosome staining (with Giemsa)

There are various techniques to stain chromosomes and achieve different types of banding. Here we mention some of them:

1. Giemsa staining:

Giemsa is a visible light dye, which binds DNA through intercalation. Visible light dyes are more stable and capable of producing clearer bands than fluorochromes. Giemsa stain is a mixture of cationic thiazine dyes and anionic eosin dyes such as eosin Y.

Positive thiazine dye molecules are smaller and two molecules of the same quickly intercalate into the negative DNA molecule, and stains it blue. The anionic eosin molecule then binds the two thiazine molecule and stains the DNA purple. Giemsa stains the hydrophobic regions better.

There are four different types of banding techniques, which can be done using Giemsa: G-bands, R-bands, C-bands & Tbanding. In each of the above mentioned staining techniques, Giemsa stain stains different regions due to difference in the pretreatment. As we all know, the histone proteins are uniformly spread through the length of the chromosomes. The non-histone proteins are spread at variable sites and responsible for loose or condensed state of different regions of the chromosomes. The loosely packed regions or euchromatin regions and tightly packed region are the heterochromatin regions. The pre-treatment processes differentially extract these proteins resulting into differently stained regions.

This is the most commonly used banding method for cytogenetic analysis using Giemsa stain. The technique was first developed by Dr. Marina Seabright in 1971.

Fig 3: Dr. Marina Seabright

(Image Source and more information on Dr. Marina Seabright here)

The cells are arrested in metaphase, swollen (made turgid), fixed, dropped and bursted. Then the chromosome spreads are air dried and chromosomes are pretreated before Giemsa staining (Fig 2 in details in previous post).

Pretreatment:

During the standard G-banding the chromosomes are mildly treated with proteolytic enzymes (trypsin) before staining with Giemsa. Standard protocol G-band staining when followed gives around 400 and 600 bands to be seen on metaphase chromosomes.

The two types of bands which are observed are

• Positive G-bands:

Positive G-bands are the darkly stained bands. These regions are hydrophobic, and favour the formation of the thiazine-eosin precipitate. The hydrophobicity is due to the hydrophobic proteins. The proteins are difficult to extract as they have more of disulfide cross-links (Fig 4). These proteins keep the regions more condensed . They form the late replicating heterochromatin and are generally AT-rich region.

Fig 4: Disulphide bond and sulfhydril group

• Negative G-bands:

The lightly stained bands are called negative G-bands. These regions are rich in GC base pairs. These are early replicating euchromatin and are less condensed. The proteins that bind these regions have more of sulfhydrils (fig 4) and are easily removed during pretreatment. These regions are less hydrophobic and less favorable for the formation of the thiazine-eosin precipitate.

Fig 5: Normal Human (Female) Karyotype. (Source: Thirumulu Ponnuraj, Kannan. (2011). Cytogenetic Techniques in Diagnosing Genetic Disorders)

This banding technique reveals the GC-rich euchromatin and produces positive bands that correspond to the negative G-bands and vice versa. This gives results reverse of the standard G-banding.

In this technique, banding is produced by metaphase chromosomes in hot phosphate buffer (

87°C) before staining with giemsa stain. The incubation causes the denaturation of the AT regions of the chromosomes because of the low melting point of these regions (

65°C) as compared to that of the GC regions (

R-banding is helps analyse the structure of chromosome ends, which stain light with G-banding but darker with R-Banding.

Fig 6: Chromosome 1 : G-banding, diagram and R-banding – (Image Source: Claude Léonard, Jean-Loup Huret Atlas of Genetics and Cytogenetics in Oncology and Haematology)

C-banding stains constitutive heterochromatin which is present around the centromeres of all human chromosomes, and is most abundant around the centromeres of human chromosomes 1, 9, 16 and the distal long arm of the Y-chromosome.

Pre-treatment:

The pretreatment involves three successive steps treatment with acid (HCl), followed by a alkaline treatment (barium hydroxide) and finally treatment with hot salts [saline- sodium citrate (SSC)]. Treatment with acid (HCl) brings about the removal of the purines. The alkaline treatment (barium hydroxide or Sodium borohydride) reduces the apurinated sugars. Chain breakage of the depurinated sites occur during treatment with hot salts solution [60°C, saline- sodium citrate (SSC)]. In this final treatment, the sites with highly repetitive sequence resist the breakage and get renatured. Also the sites with proteins having strong interaction are protected. Therefore, the sites protected by protein or having highly repetitive sequences like centromeres get stained.

Fig 7: (a) the G-banded karyotype of a male Bradypus torquatus (2n = 50) (b) C-banded karyotype of a male Bradypus torquatus. (Image Source: Azevedo N et. al. (2012). BMC evolutionary biology. 12. 36.)

T-banding involves the staining of telomeric regions of chromosomes. The chromosomes (slides) are incubated in a phosphate or PBS buffer at 87°C followed by staining with Giemsa solution or acridine orange(OA) . T-bands are heat-resistant regions, particularly rich in C-G pairs. They make up around 15% of all the bands but contain around 65% of all the genes mapped.

Fig 8: T banding

Banding techniques using other stains:

Quinacrine mustard is an alkylating agent which fluoresces brightly under UV light. These bands are visible under a fluorescence microscope. The alternating bands of bright and dull fluorescence are called Q bands. The bright bands are AT rich region and the dull bands are GC rich region (similar to G banding). Q bands are useful in distinguishing the human Y chromosome and various chromosome polymorphisms involving satellites and centromeres of specific chromosomes.

Fig 9: Q-banding

NOR banding

NOR banding involves the staining of “nucleolar organizing region” by silver stain (silver nitrate solution).The NOR contains rRNA genes. It is thought that the silver nitrate attaches to the nucleolar proteins and not the rDNA in itself. In humans, the NORs are found on the short arms of the chromosomes 13, 14, 15, 21 and 22, the genes RNR1, RNR2, RNR3, RNR4, and RNR5 respectively. They code for 5.8S, 18S, and 28S rRNA. The silver stain usually stains the transcriptionally active rRNA genes. Changes in the NOR number and size help explain changes in transcriptional activity in different environment and conditions.

Fig 10: Stained NORs

(Just for info:Read how banding technique allows to find diversification in the species of fishes

DAPI/Distamycin A Staining:

This is a fluorescent staining technique for labelling a specific subset of C bands. DAPI/Distamycin A staining is used in identification of peri-centromeric breakpoints in chromosomal rearrangements and chromosomes that are too small for standard banding techniques.

4′-6-diamidino-2-phenylindole (DAPI) is a DNA-binding AT-specific fluorochrome. which gives blue fluorocence. Distamycin A is an DNA-binding AT-specific oligopeptide antibiotic. Distamycin A pretreatment results in decrease in fluorescence by DAPI staining, allowing only some specific regions of constitutive heterochromatin to brightly fluoresce.

Fig 11: Comparison between only DAPI and DAPI/Distamycin A staining.

These bright bands include the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y.

Hence different banding technique help in staining certain regions darkly or brightly . These regions can be compared with that in homologous chromosomes or chromosomes of different individuals or species to obtain information on diseases, evolution and parentage.

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(Image sources: Fig 8: T banding Fig 9: Q banding Fig 10: Stained NORs Fig 11: DAPI (Thermofisher) and DAPI/Distamycin A staining)


What is the Giemsa Stain? (with pictures)

Giemsa stain is a standardized mixture of dyes that makes different cell types stand out clearly in a blood smear or thin slice of tissue. This stain is named for the German chemist, Gustav Giemsa, who first developed it for his work in studying the parasite that causes malaria — Plasmodium. In order to ensure that the technician who examines the sample can obtain an accurate reading, the steps of the staining procedure must be standardized as well as the mixture of dyes. Giemsa stain is called a differential stain because it produces different colors depending on what it bonds to, such as cytoplasm or DNA.

The formula for Giemsa stain has been adjusted over time to improve the stability of the dyes and the colors that result. Current standard mixtures include methylene blue, eosin, and sometimes azure B. These dyes are often stored in a dry powdered form and mixed with water just before they are used. If water is present in the dye mixture before it is used, some of the compounds will oxidize and stain incorrectly.

The exact steps of the procedure for using Giemsa stain may vary depending on which organism or cell type the sample is being examined for as well as the composition of the sample itself. A sample that will be stained using the Giemsa stain is usually smeared on, or affixed to, a slide very soon after it is collected. A thin blood smear is generally fixed by being dipped in methanol, while a thick blood smear is simply allowed to dry completely at room temperature. The slide is then soaked in the stain for a set amount of time and then rinsed with water that has a neutral pH. Slides are allowed to air dry before viewing.

Due to the differential staining produced by Giemsa stain,Plasmodium cytoplasm stains light blue while the DNA appears red or purple. Another parasite, Giardia lamblia, is tinted pink-purple except for the DNA, which stains very dark blue. Histoplasma capsulatum, a fungus, is found in its yeast form in human white blood cells and stains dark blue.

This staining process is also helpful in chromosome studies and in visualizing the differences between various blood cells. A chromosome stains very dark blue in some sections and light blue in others. This causes a banding effect that helps geneticists find places where the chromosomes have gone through unusual changes. Red blood cells stain pink, while the granules in mast cells show up as purple specks. White blood cells stain various shades of blue, allowing the different types — basophils, eosinophils, neutrophils, and others — to be distinguished from one another.


What is Chromosome Banding? (with pictures)

Chromosome banding is the transverse bands that appear on chromosomes as a result of various differential staining techniques. Differential stains impart colors to tissues, so that they may be studied under a microscope. Chromosomes are thread-like structures of long deoxyribonucleic acid (DNA) filaments, which coil into a double helix and are made up of genetic information, or genes, that are arranged in a crosswise manner down the length.

To analyze chromosomes under a microscope, they need to be stained when they are undergoing cell division during the meiosis or mitosis. Mitosis and meiosis are cell division processes that are divided into four phases. Those phases are prophase, metaphase, anaphase, and telophase.

Crytogenetics is the study of the function of cells, the structure of cells, DNA, and chromosomes. It employs various techniques for staining chromosomes, like G-banding, R-banding, C-banding, Q-banding, and T-banding. Each staining technique allows scientists to study different aspects of chromosome banding patterns.

Giemsa banding, also known as G-banding, enables scientists to study chromosomes in the metaphase stage of mitosis. Metaphase is the second stage of mitosis. At this phase the chromosomes are lined up and attached at the centers or their centromeres, and each chromosome appears in an X shape form.

Before applying stain to the chromosomes, they must first be treated with trypsin, which is a digestive fluid found in many animals. The trypsin will start to digest the chromosomes, allowing them to better receive the Giemsa stain. Giemsa stain was discovered by Gustav Giemsa, and is a mixture of methylene blue and the red acidic dye, eosin. Q-banding uses quinicrine, which is a mustard type solution. It produces results that are very similar to Giemsa, but has fluorescent qualities.

DNA is made up of four base acids that appear in pairs — adenine paired with thymine, and cytosine with guanine. Giemsa stain creates chromosome banding patterns with dark areas rich in adenine and thymine. The light areas are rich with guanine and cytosine. These areas replicate early and are euchromatic. Euchromatic is a genetically active area that stains very lightly with dye treatments.

Reverse-banding, or R-banding, produces chromosome banding patterns that are the opposite of G-banding. The darker areas are rich with guanine and cytosine. It also prodcues euchromatic parts with high concentrations of adenine and thymine.

With C-banding, the Giemsa stain is used to study the constitutive heterochromatin and the centromere of a chromosome. Constitutive heterochromatins are areas near the center of the chromosome that contain highly condensed DNA that tend to be transcriptionally silent. The centromere is the region at the very center of the chromosome.

T-banding allows scientists to study the telomeres of a chromosome. The telomeres are the caps that are on the each of the chromosomes. They contain repetitive DNA and are meant to prevent any deterioration from occurring.

Once the chromosomes are stained with Giemsa, researchers can clearly see the alternating dark and light chromosome banding patterns that are produced. By counting the number of bands, the karyotype of a cell can be determined. The karyotype is the characterization of chromosomes for a species according to size, type, and number.


Contents

It is specific for the phosphate groups of DNA and attaches itself to regions of DNA where there are high amounts of adenine-thymine bonding. Giemsa stain is used in Giemsa banding, commonly called G-banding, to stain chromosomes and often used to create a karyogram (chromosome map). It can identify chromosomal aberrations such as translocations and rearrangements. [ citation needed ]

It stains the trophozoite Trichomonas vaginalis, which presents with greenish discharge and motile cells on wet prep. [ citation needed ]

Giemsa stain is also a differential stain, such as when it is combined with Wright stain to form Wright-Giemsa stain. It can be used to study the adherence of pathogenic bacteria to human cells. It differentially stains human and bacterial cells purple and pink respectively. It can be used for histopathological diagnosis of the Plasmodium species that cause malaria [2] and some other spirochete and protozoan blood parasites. It is also used in Wolbachia cell stain in Drosophila melanogaster. [ citation needed ]

Giemsa stain is a classic blood film stain for peripheral blood smears and bone marrow specimens. Erythrocytes stain pink, platelets show a light pale pink, lymphocyte cytoplasm stains sky blue, monocyte cytoplasm stains pale blue, and leukocyte nuclear chromatin stains magenta. It is also used to visualize the classic "safety pin" shape in Yersinia pestis.

Giemsa stain is also used to visualize chromosomes. This is particularly relevant for detection of Cytomegalovirus infection, where the classical finding would be an "owl-eye" viral inclusion. [3]

Giemsa stains the fungus Histoplasma, Chlamydia bacteria, and can be used to identify mast cells. [4]

Giemsa's solution is a mixture of methylene blue, eosin, and Azure B. The stain is usually prepared from commercially available Giemsa powder.

A thin film of the specimen on a microscope slide is fixed in pure methanol for 30 seconds, by immersing it or by putting a few drops of methanol on the slide. The slide is immersed in a freshly prepared 5% Giemsa stain solution for 20–30 minutes (in emergencies 5–10 minutes in 10% solution can be used), then flushed with tap water and left to dry. [5]


Sequence organization was revealing by heating DNA to a single-stranded state then allowing the DNA to reanneal by cooling.
This revealed several types of repetitive DNA.
Multiple "copies" of similar functional repetitive sequences can be described as dispersed gene families (globin genes, actins, tubulins).
Non-functional copies of genes are known as pseudogenes.
Tandem gene family arrays are made up of multiple copies of the same gene all next to each other (such as histones).
The nucleolar organizer, which is cytologically distinct, is a tandem array of genes that encode ribosomal RNA.
Noncoding functional sequences, such as the short tandem repeats that act to maintain the telomeres at the ends of a linear chromosome.

There are a number of sequences with no known function include
1) Highly repetitive centromeric DNA including satellite DNA.
2) Variable number tandem repeats (VNTRs) or minisatellite DNA which provide the differences in DNA used in DNA fingerprinting.
3) Microsatellites, regions of dinucleotide repeats

Transposed sequences are "jumping genes" that are dispersed throughtout the genome.
Transposons move as DNA elements and retrotransposons move via an RNA intermediate which is reverse transcribed and reinerted into the genome.
Examples of a retrotransposons include the 1-to 5 kilobase Long interspersed elements (LINES) and the much smaller (>200 basepairs) short interspersed elements (SINES)
Such as the human Alu sequences.
The presence of these various elements provides a great deal of variety to the spacing and locations of genes in the genomes of organisms.


The involvement of nucleosomes in Giemsa staining of chromosomes. A new hypothesis on the banding mechanism

A new hypothesis is proposed on the involvement of nucleosomes in Giemsa banding of chromosomes. Giemsa staining as well as the concomitant swelling can be explained as an insertion of the triple charged hydrophobic dye complex between the negatively-charged super-coiled helical DNA and the denatured histone cores of the nucleosomes still present in the fixed chromosomes. New cytochemical data and recent results from biochemical literature on nucleosomes are presented in support of this hypothesis. Chromosomes are stained by the Giemsa procedure in a purple (magenta) colour. Giemsa staining of DNA and histone (isolated or in a simple mixture) in model experiments results in different colours, indicating that a higher order configuration of these chromosomal components lies at the basis of the Giemsa method. Cytophotometry of Giemsa dye absorbance of chromosomes shows that the banding in the case of saline pretreatment is due to a relative absence of the complex in the faintly coloured bands (interbands). Pretreatment with trypsin results in an increase in Giemsa dye uptake in the stained bands. Cytophotometric measurements of free phosphate groups before and after pretreatment with saline, reveal a blocking of about half of the free phosphate groups indicating that a substantial number of free amino groups is still present in the fixed chromosomes. Glutaraldehyde treatment inhibited Giemsa-banding irreversibly while the formaldehyde-induced disappearance of the bands could be restored by a washing procedure. These results correlate with those of biochemical nucleosome studies using the same aldehydes. Based on these findings and on the known properties of nucleosomes, a mechanism is proposed that explains the collapse of the chromosome structure when fixed chromosomes are transferred to aqueous buffer solutions. During homogeneous Giemsa staining reswelling of the unpretreated chromosome is explained by insertion of the hydrophobic Giemsa complex between the hydrophobic nucleosome cores and the superhelix DNA. Selective Giemsa staining of the AT-enriched bands after saline pretreatment is thought to be due to the, biochemically well-documented, higher affinity of arginine-rich proteins present in the core histones for GC-enriched DNA, which prevents the insertion of the Giemsa complex in the interbands. Production of Giemsa bands by trypsin pretreatment can be related to the action of this enzyme on the H1 histones and subsequent charge rearrangements.(ABSTRACT TRUNCATED AT 400 WORDS)


Giemsa banding (GTG, GTW, GAG, GTL)

The introduction of Giemsa banding (G‐bands) in 1971 by Sumner et al. was another major advance in the field of cytogenetics. It eliminated the need for an expensive fluorescence photomicroscope and provided permanently stained slides with very high‐resolution bands. Today, Giemsa banding (or similar Romanowsky dyes) is the most widely utilized staining technique for chromosome analysis.

In human chromosomes, the 30‐nm fiber of DNA forms loops of approximately 75–100 kb, which are tethered or anchored at their bases to form what is known as a chromosome scaffold. This structure can be seen by removing most histones and non-histones from the chromosomes and surface‐spreading them appropriately for electron microscopy. The residual structure appears as a scaffold, with numerous and extensive loops of DNA radiating from a coarsely fibrous structure that resembles a metaphase chromosome. The final packaging of the 30‐nm fiber of DNA into chromosomes results in a 10,000‐fold reduction in DNA length. Fixation in 3 : 1 methanol‐acetic acid is an important preliminary step for G‐banding. Fixation extracts a portion of all histones, especially H1, and a group of non-histones in the 50,000‐ to 70,000‐dalton (Da) range, but it is far from a complete removal of proteins. The crosslinking data indicated that the conformation of chromosomal proteins in relation to DNA strongly influences banding.

Giemsa stain is a complex mixture of dyes that may vary in concentration, purity, and ratio. The main components are the basic aminophenothiazine dyes – azure A, azure B, azure C, thionin, and methylene blue‐and the acidic dye, eosin. The thiazin dyes vary in the number of methyl groups attached to a core of two benzene rings bound together by nitrogen and a sulfur atom. Several studies have been done concerning the band‐producing ability of the individual components of Giemsa stain. Methylene blue or any of the azures alone produces banding.

Chromatin is divided into two main groups: heterochromatin and euchromatin. Constitutive heterochromatin is highly condensed, very repetitive, and transcriptionally inactive during interphase, in order for gene transcription to occur. One type of euchromatin, facultative heterochromatin, behaves like heterochromatin in a developmentally controlled manner, being transcriptionally inactive, late replicating, and condensed during interphase. The inactive X chromosome in the somatic cells of female mammals is an example of facultative heterochromatin.

G banding requires methanol/acetic acid‐fixed cells spread onto slides cytoplasmic background interferes with good G‐bands, so proper cell dilution and spreading is essential. Slides are sufficiently dried (for 2–4 days at room temperature), or baked at low temperatures (e.g., 60 °C) for 2–18 hours, or high temperatures (e.g., 90–95 °C) for 20–60 minutes to dehydrate them (see Table 6.4). Slides that are insufficiently “aged” by natural means or by heating will not respond at all to the trypsin, no matter how long it is applied, and the chromosomes will appear unbanded.

Trypsin exposure depends on its brand, concentration, and temperature, but may also be affected by preparation and working conditions. Recrystallized forms have a higher level of activity, thereby requiring less time or a lower concentration. A stock trypsin solution can be prepared in advance, and small aliquots can be frozen until needed. At lower temperatures, the working solution has decreased activity. Higher pH (approximately 8) and higher temperature increase the enzymatic action of the trypsin and may be useful with cells that appear to be resistant to standard concentrations. Exposing slides to trypsin in a vertical fashion (rather than horizontal) may enhance its activity slightly.

A fetal bovine serum rinse is helpful in stopping the action of the trypsin. Serum contains alpha‐1‐antitrypsin, which inhibits the trypsin by complexing it with the protein in the serum. Other rinses that are commonly used include pH 8.0 buffer, 0.85% (normal) saline, Hanks’ balanced salt solution (HBSS), and 70% ethanol.

Determination of G‐banded chromosome resolution

As a rule, the better the banding resolution is, the better will be the chance of finding a small chromosome abnormality. This rule is more reliable for deletions and duplications than for centromere displacement (inversions, neocentromeres) because the constriction of the centromere becomes more difficult to visualize as the chromosomes become longer and thinner. However, there is a need to quantify the banding resolution of a given cell (on the analysis sheet) or chromosome study (on the final report) so that the limits of resolution can be inferred. There are several methods for determination of band levels.

The Vancouver method involves counting bands in segments for some chromosomes (e.g., chromosomes 1, 11, and 12) and all bands in others (chromosomes 10 and X). Count only G‐positive bands and do not count the centromere. Total the bands then look at a chart that has increments of 50 bands at the lower range and 100 bands at the higher end to determine haploid band resolution.

ChromosomeResolution 350Resolution 450Resolution 550Resolution 850
1p31-p321133
10551219
11p2256
12q45814
X681218
Total18214060
Haploid band resolution determination by the Vancouver method

Johnson and Stallard method

Johnson and Stallard used a method that quantifies the number of light and dark G‐bands present on one chromosome 10. Some laboratories account for a difference in resolution between the two chromosome 10 homologues in a cell by counting both homologues and dividing the total by 2 before using the haploid parameters.

No of bands presentHaploid banding level
12375
13-14400
15-16425
17-18450
19-21475
22-23500
24-25525
26-28550
29575
30600
31625
32650
33675
34700
35725
36750
37775
38800
39825
40-41850
Haploid band resolution determination using chromosome 10 by the Johnson and Stallard method

Welborn method

The Welborn method involves counting all dark and light bands on one homologue each of chromosomes 1 and 2, counting the centromere as a band in each arm. Total the bands from both chromosomes and then multiply by 6 to get the haploid band resolution, since chromosomes 1 and 2 represent 1/6 of the genome.


Watch the video: Oberflächliche Unterscheidung von Blutkörperchen unter Giemsa-Färbung (May 2022).