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A variety of glassware like burettes, pipettes, measuring cylinders, flasks, separating funnel etc. are used for experimental purposes in a laboratory. All of these need to be used and handled correctly, otherwise the chances of experimental error and damage to the glassware increase significantly. Being made of glass, they need special care while washing and using in experiments because even a minor mistake in handling of glassware may result in breakage of the apparatus. With this precautionary note, let us discuss about some of the specific glassware used in the laboratories.

Burette

Burette is a long graduated glass tube with a stopcock at one end for delivering a known/desired volume of a liquid, especially in titration experiments. It contains a stopcock at the lower end and a tapered capillary tube as the stopcock’s outlet. The flow rate of liquid from the tub stopcock’s outlet can be controlled with the help of stopcock valve. Burette is used for measuring the volume of a solution/chemical added in a quantitative (titrimetric) estimation experiment.

Procedure of using a Burette

The burette reading must be noted before and after delivering the chemical/ solution from the burette. The difference in these two readings is the volume of the chemical/solution released out of the burette. The solution should be released slowly/drop-by-drop to avoid any experimental error/mistake. If the solution/chemical is allowed to pour out too fast, the wall of the burette will not drain properly and some liquid may remain sticking to the surface of the walls. Moreover, excessive amount of chemical may be released/dropped during the experiment, particularly during the titration experiment wherein we need to know the exact amount of acid/base needed to get neutralization point. Thus, any minor mistake may lead to get a faulty reading. The measuring capacity of a burette usually used in a laboratory is 50 mL.

Introduction

A variety of glassware, plasticware, and other apparatus are used for experimental purposes in a laboratory. They need to be washed, cleaned, dried, and sometimes sterilized before they can be used in the next set of experiments. Glassware needs special care of handling during washing, drying, sterilization, etc. because even a minor mistake in handling of glassware may result in its cracking/breakage and it may become unserviceable. All of these may result in a delay in experimentation if spare piece of the glassware is not available or it may affect serviceability of the associated apparatus.

A large number of glassware such as beaker, flask, measuring cylinder, burette, test tube, etc. are regularly used in a laboratory, which requires careful handling during the different stages of experiment as well as after completion of the experiment while preparing them for another set of experimentation.

Glass being a complex silicate, its properties depends on the type of silicate anions in the structure and the cations formed. Thermal properties of glass changes by the addition of boron oxide (B2 O3 ); hence, borosilicate glass is extensively used in the laboratories. With this brief background information, let us discuss about the necessity, procedures, and precautions during washing, cleaning, drying and sterilization of laboratory-wares. Figure 3.1 shows various advantages of using borosilicate glass in a laboratory.

Good laboratory practices require clean glassware, because an experiment may give erroneous result if dirty glassware is used at any step of the experiment. Hence, the glassware must be physically and chemically clean; and in many cases, it must be microbiologically clean or sterile. The commonly used glassware in a laboratory include test tube, measuring cylinder, pipettes, beakers, flasks, microscopic slides, etc.

Introduction

While experimenting with chemicals, sometimes we need to dry the chemical/ solution (solid, liquid or gas) using a process like evaporation or distillation, mainly to remove water or different solvents. Drying is the process of evaporation or distillation to remove water/solvent from a solution, solid or even from gas. Generally, a drying agent (usually an anhydrous inorganic salt) is used for this process, which reacts with the water (present in the chemical/ solvent to be dried) to form a hydrate. For example, anhydrous MgSO4 is one of the drying agents. The drying agents are recognized by their capacity (the rate at which they absorb water) and their intensity (the amount of water left behind in the solvent at equilibrium). No chemical reaction or change in phase occurs during the drying process.

Type of Solvents

Apart from water (a universal solvent), there are three families of organic solvents used in laboratories.

1) Oxygenated solvents: Alcohols, Ketones, Glycol ethers, Glycol esters, etc.

2) Hydrocarbon solvents: Aliphatic and Aromatic hydrocarbons.

3) Halogenated solvents: Chlorinated hydrocarbons.

 

Introduction

For proper and efficient experiment to be conducted, experimenters need so many chemicals to deal with. This includes different acids, base, salt, different kind of indicators and so on. Some of these chemicals are safe for health, but some of them are dangerous to health when come in close proximity to our body while some of the chemicals cause health issues on inhaling. These may cause improper functioning of some of our physiological activities. Acids and bases are very dangerous for our tissues when come in our contact. These may cause serious respiratory problem in addition to injuries on coming to our contact. Some chemicals are exploratory yet they may cause even death of the person working with them in laboratory. Therefore, mere use of the chemicals in laboratory is not only the objective of the experimenter. The person working in laboratory should also be concerned about the proper use and handing of the chemical while working with it. Thus, every researcher of an institution who has to work with chemicals in a laboratory should be made aware about the chemicals to be used in the laboratory and the consequences if any mishandling of a laboratory chemical occurs. Not only these, suitable precautionary measures should also be known to the researcher along with ensuring the availability of the required safety equipment/installations. Hence, a proper understanding of the ways of handling of chemicals in a laboratory is of prime importance for the personals like you who are working or have to work in laboratory.

Safe Handling of Chemicals in Laboratory

Safe use/handling of chemicals in laboratory include minimized exposure to chemicals, maintaining safety data sheets, proper labelling, storage, segregation, and transport of chemicals for personal hygiene.

A

Adenocarcinomas 3, 7

Adenomas 7

ALDH1 17, 18

Anaplastic carcinomas 7

B

Babesiosis 1

Bcl 17, 21, 23

Benign tumors 3, 5

Biomarkers 17, , 29, 30, 31

BRCA 31

C

Canine 1, 2, 5, 7, 8, 9, 10, 11, 12, 13, 17, 19,

19, 22, 23, 24, 27, 29, 30, 32, 33, 34

Canine distemper 1

Canine Mammary Tumors 1, , 5, 7, 8, 13, 17,

19, 29, 32, 33

Canine parvoviral diarrhoea 1

Carcinogenesis 10, 25

CHEK2 15

Classification and Grading 7

Companion animals 1, 9

COX inhibitors 21, 26

Cyclooxygenases 23, 26, 35

D

Deracoxib 21, 26

Diagnosis 19, 20

Doxorubicin 21

E

EMT 17, 25

Epidemiology 11

Estrogen receptors 20

FGFR2 15

Agostini M., Enzo M. V., Bedin C., Belardinelli V., Goldin E., Del Bianco P., and Nitti D.

(2012). Circulating cell-free DNA: a promising marker of regional lymphnode metastasis

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Badowska-Kozakiewicz A. M., and Malicka E. (2012). Immunohistochemical evaluation of

expression of heat shock proteins HSP70 and HSP90 in mammary gland neoplasms in

bitches. Polish Journal of Veterinary Sciences 15 (2), 115-18.

Beffagna G., Sammarco A., Bedin C., Romualdi C., Mainenti M., MolloA. and Zappulli V.

(2017). Circulating cell-free DNA in dogs with mammary tumors: short and long fragments

and integrity index. PLoS One 12 (1), e0169454.

Birdi R., Kumar B. S., Gupta K., Kashyap N., & KumarA. (2019). Circulating level of heat

shock protein 27 is elevated in dogs with mammary tumors. 3 Biotech 9 (6), 1-9.

Campos L. C., Lavalle G. E., Estrela-Lima A., Melgaço de Faria J. C., Guimaraes J. E., DutraA.

P. and Cassali G. D. (2012). CA 15.3, CEA and LDH in dogs with malignant mammary

tumors. Journal of Veterinary Internal Medicine, 26 (6), 1383-88.

Mammary tumors are the most common type of neoplasia found in female dogs with high percentage of malignancy and rate of mortality. The rate of incidence of these mammary neoplasia and dysplasia is continuously advancing nationally and internationally over the last 45 years. There are different risk factors such as age, diet, hormonal exposure and breeds, which are required to be monitored in order to control the incidence of CMTs. In addition to conventional therapies such as surgical excision, radiotherapy and chemotherapy, additional precision therapeutic agents targeting TKIs, p53 tumor suppressor gene, cyclooxygenases, epithelial-mesenchymal transitions and various hormones/ hormonal receptors could be beneficial for the treatment of CMTs. Scientific efforts are also being made to develop different biomarker panels and tests for prognosis/diagnosis of the diseases as well as to develop vaccines against CMTs.

In GADVASU, under the DBT sponsored project on “Development and evaluation of serodiagnostic assay for timely diagnosis and prognosis of mammary tumors”, we have generated and used hyper-immune sera raised against certain canine recombinant mammary tumor associated proteins for the standardization of sandwich and indirect ELISA for serodiagnosis of canine mammary tumor. The sensitivity and specificities of the assay’s ranges between 85-88% and 80-85%, respectively. However, an antibody against a single marker when used for different histotypes of CMT, its specificity reduced significantly. Multiplex assays are capable to detect many specific antigens in a single run whereas a traditional ELISA is used only to detect a single antigen. It has high-throughput potential, providing more results per sample, with lower sample volumes and with utmost sensitivity and specificity. Attempts are being made to increase the sensitivity and specificities of the assays by multiplexing various markers together (Fig.4).

Although there have been multiple biomarkers for mammary tumors both in human and canines reported over the time (Table 3), but no absolute biomarker for diagnosis and prognosis of CMT has been found yet. Out of several studies on clinical applications of different mammary tumor biomarkers, some showed a particular promising biomarker, on the other hand controversial results also existed for the same. For example, HER-2, which is one of the most prominent bio-marker both in HBC and CMT. HER-2 overexpression is associated with poor prognosis and reduced overall survival of the subject, but there are studies available which suggest no difference in HER-2 expression between benign and malignant tumors and studies showing HER-2 expression suggesting prolonged survival (Kaszak et al., 2018). Various biomarkers of mammary tumors are not mammary tumor specific such as p53, COX-2, PCNA etc. These markers undergo alterations in their expression level in all tumors. By keeping this in mind we need to device a panel of different markers to ensure the correct prognosis and diagnosis of canine mammary tumors. There are limited studies and only few commercial kits (for human) available using such combinations (Table 4).

One of the most promising areas of veterinary research these days is cancer immunotherapy and the development of CMT vaccines. DNA vaccines, also known as gene vaccines, are designed to transfer functional genes to target cells, allowing them to express functional proteins. Because endonucleases may easily access bare DNA, it requires a reliable and safe delivery route, which might be biological or non-biological. Different delivery strategies have been tried in human breast tumors to transport tumor antigens such Mucin-1 (MUC-1), Twist, HER-2, mammaglobin-1 and p53, as well as immunostimulatory molecules like IL-12 and IL-6. There have been various studies/ trials in canine animals using different molecules like IL-12, HER-2 and IFN-γ as vaccine candidates, with no clinically relevant outcomes or longlasting cell mediated and humoral immune response outcomes

In addition to the conventional treatment methods i.e., surgery, chemotherapy and radiotherapy, there are some adjuvant targeted therapies which block the growth of cancer by altering the individually expressed specific molecules responsible for tumor cell proliferation, survival, metastasis or microenvironment. The majorly studied adjuvant therapies include use of hormones, tyrosine kinase receptors, anti-cyclooxygenases, anti-tumor suppressor gene p53 and epithelial-mesenchymal transition inhibition.

The main aim of hormonal therapy (Table 2) is to inhibit the hormone induced stimulation of carcinoma cells, which could be achieved either by ovariohysterectomy or different treatments like use of specific estrogen receptor modulators (SERMS), aromatase inhibitors for suppression of estrogen synthesis etc. A commonly used estrogen receptor (ER) antagonist drug tamoxifen for treatment of ER positive breast cancer in women, has also been tested against CMTs. However, it is not a viable treatment option in dogs due to associated side effects. Ovariohysterectomy is considered as a better and practical solution. The use of hormonal therapy in CMTs is based on the evidence of hormone dependency of these tumors as well as lessons learnt from hormonal therapy outcomes from human breast cancer. This therapy significantly ameliorates the recurrence of breast cancers and prolongs the survival rates in women having breast cancers positive for hormone receptors. However, the results of the canine investigations, which are all retrospective and non-randomized, are contradictory. Detailed research and prospective randomized trials, where effect of OHE is studied in context of hormone receptor status, are required to determine the role of hormonal therapy in CMTs.

Surgical removal is the routine treatment method for mammary tumors, however, there is no consensus on the optimal procedure. There are supporting factors for every surgical procedure conducted for mammary tumors including lumpectomy (removal of tumor only), mastectomy (affected gland removal only), radical mastectomy (complete mammary gland and associated lymphatic tissue removal) and modified radical mastectomy (removal of complete mammary gland, lymph nodes and associated chest muscles).The more extensive procedures do not increase survival in dogs when compared to the other procedures, and the benefits of the simpler procedures are clear. Further, the radical mastectomy increases the disease-free interval but not the survival duration in cats.

A thorough history with complete physical examination including careful palpation of the mammary glands should be performed. As there are chances that dogs may be suffering from any other systemic ailments during middle to old age. In dogs with multiple tumors, all tumors should be excised and their location and size should be noted. Histopathological examination of the excised tumors should be carried out to classify mammary gland tumors. Mammary tumors are classified based on some of the parameters i.e., tumor size, lymph nodes, metastasis. Therefore, to stage a dog with mammary tumors information of these parameters needs to be collected and recorded. If lymph nodes are palpable and/or clinically enlarged, fine-needle aspiration should be performed for cytological evaluation. The technique is less invasive but with higher accuracy. Thoracic radiographs remain the mainstay for diagnosing the metastatic condition. The mammary tumors most commonly metastasize into lungs in dogs. The staging of CMTs becomes necessary as it is likely to confer some prognostic significance and provide guidance to veterinarians to make treatment decisions.

There are countless biomarkers present in cells related to and determining the progress of a disease. Identifying these markers could aid in detecting cells with metastatic potential (Ramaswamy et al., 2003).

The elevation of cyclo-oxygenase (Cox-2) expression was observed in CMT cases through enzyme immunoassay (Queiroga et al., 2005). In further study, enhanced expression of BRAC1and BRAC2 genes, certain enzymes such as matrix metalloproteinases (MMP-2 and MMP-9) by immunohistochemical examinations, and Bcl-2 & Bcl-XL elevated level in dogs with mammary neoplasm have been reported by Queiroga et al. (2011b).

6.1 Age

Age is one of the most important risk factors for developing mammary tumors in dogs. A comprehensive study carried on dogs has found that the risk of mammary tumors become significant by 8 years of age and increases linearly thereafter. The age of onset of tumors, however, may vary depending on the life span of the dog breed. An average age of CMT onset is 8-11 years, indicating as a condition of the middle-aged or older dogs. Mammary tumors are generally rare in dogs under 3 years of age, unless the dogs have been given exogenous hormones such as progestins

6.2 Breed

Most consistently listed breeds in which mammary tumor occur include, English springer spaniel, Brittany, Cocker spaniel, English setter, Pointer, Afghan hound and German shepherd, in addition to the smaller breeds such as Poodle, Maltese, Chihuahua, Beagle, Dachshund and Terriers. These studies clearly show that the risk for mammary tumors has some breed association and, therefore, having a genetic component. Pure breeds of dogs are generally at a greater risk i.e., known to account for 80% of the submitted cases. Some studies have further shown that certain lines within specific breeds are at more risk than others.

The incidence of mammary tumor accounts for 25% of all the recorded tumors in dogs worldwide. Overall, about 198 cases of CMT are recorded per one lakh female dogs annually throughout the world which is quite an alarming percentage (Gray et al., 2020). Malignancy has also been recorded up to 45% which increases the fatality among morbid cases. Epidemiology of CMT is poorly documented particularly in India. In an epidemiological study conducted on canine tumors in Jammu in 2018, the overall prevalence was only 2% over a period of one year. The malignant tumors accounted 41.66% of the mammary tumors (Sharma et al., 2018). The rate of benign and malignant tumors incidence was reported as 46% and 54%, respectively. In another study conducted on the incidence of CMT in Ludhiana (Punjab) in 2020, it was found that 33.96% tumors were benign and 66.03% were malignant (Dash et al., 2020).

• General considerations

 • Polyacrylamide gel electrophoresis (PAGE)

 • SDS-polyacrylamide gel electrophoresis (SDS PAGE)

• Nucleic acid hybridization

 • Radio labelled probes

 • Biotin labelled probes

 • DNA finger printing

 • Polymerase chain reaction (PCR)

 • Reverse transcriptase polymerase chain reaction (RT PCR)

• General considerations

 • Indications

 • Limitations

 • Techniques of bone marrow examination

 • Cell types

 • Cellular composition of bone marrow

 • Morphology of the cells

 • Interpretation of bone marrow examination

• General considerations

 • Blood glucose

• Total serum protein

 • Serum albumin

 • Serum globulin

 • Serum gamma globulin

 • Serum calcium

 • Serum inorganic phosphorus

 • Serum magnesium

 • Serum cholesterol

 • Serum creatinine

 • Serum urea

 • Serum chloride

 • Serum sodium

 • Serum potassium

 • Serum Glutamic Oxaloacetic Transaminase

 • Serum Glutamic Pyruvic Transaminase

 • Serum alkaline phosphatase

• General considerations

 • Collection of blood

 • Storage

 • Examination of blood

 • Coagulation time

 • Bleeding time

 • Determination of movement of parasites under wet film

• Total erythrocyte count

 • Total leucocyte count

 • Differential leucocyte count

 • Hemoglobin

 • Erythrocyte sedimentation rate

• Hematocrit value

 • Mean corpuscular volume

 • Mean corpuscular hemoglobin

 • Mean corpuscular hemoglobin concentration

 • Erythrocyte fragility test