Laboratory Safety Guidelines PDF

## Laboratory Safety ### Introduction Precautions for working with disease agents should be followed by every microbiological laboratory to avoid the following dangers: (1) accidental infection or cross-contamination within the laboratory, which confuse experimental results, (2) spread of infectio...

## Laboratory Safety ### Introduction Precautions for working with disease agents should be followed by every microbiological laboratory to avoid the following dangers: (1) accidental infection or cross-contamination within the laboratory, which confuse experimental results, (2) spread of infection to adjacent farms, communities, or large areas, (3) infection of laboratory personnel, and (4) spread of infection through released biologics. The world literature contains references to approximately 6000 cases of accidental infection of laboratory workers and undoubtedly thousands more not reported. Many of the laboratory-acquired infections are due to zoonotic disease agents of animals transmissible to humans. ### Objectives - Practically apply the basic safety measures in virology laboratory. - Describe the danger of neglecting the safety issues in your lab practice. ### General Laboratory Safety Measures - There must be no eating, drinking, smoking, or applying any cosmetics in the laboratory. - Unauthorized persons, particularly children and infants, should not be allowed in the laboratory. - No mouth-pipetting should be permitted due to the possibility of accidental ingestion of contaminants. - Hands should be washed when coming in the laboratory, after handling cells or virus and before leaving the laboratory. - Avoid touching the eyes, nose, and mouth or face while working in the laboratory. - When gloves have been worn they should be used only for specific task and taken off when it is done. Not touching doorknobs and surfaces with contaminated gloves. - Laboratory coats should be worn when doing cell culture or virus work and should be left in the laboratory. Laboratory coats should not be worn while eating or outside of the laboratory. - All materials should be clearly labeled with the user's name and the contents therein. - All contaminated materials should be placed in decontamination pan and autoclaved prior to being washed or disposed. - Whenever working with syringes and needles, special containers (that are sealed and autoclavable) should be provided for their disposal. Do not replace the cap on the needle. Drop the uncapped syringe into the disposal container immediately after use. These containers are then autoclaved prior to disposal. - Stock solutions of suitable disinfectants should be available at each work station. Any spills should be promptly cleaned with the disinfectant. - Cages that contain infected animals should be disinfected or autoclaved and the infected animal carcasses and tissues should be properly bagged and incinerated. - Biological materials should never be taken to home from the laboratory. ## General Scheme For Viral Diagnosis - **Field Diagnosis** - Clinical Sings - Some diseases have a pathognomonic clinical sings, for example: Infectious laryngotracheitis virus (ILT) - Bloody cough - Some diseases have a pathognomonic Postmortem lesions, for example: Newcastle Disease virus (NDV) - Peticeal hemorrhage on proventricular gland tips - **Laboratory Diagnosis** - **Direct Diagnosis** (Detection of virus antigen) - Electron microscopy: have some disadvantages as: 1) expensive, 2) not available and 3) need a high virus titer about 10-10 virus unit. - Detection of virus antigen: - Cell staining: Staining of nervous tissue with Neissler's stain showing intra-cytoplasmic inclusion bodies called Negri bodies. - Serological tests: AGPT, SNT, HI, ELISA and FAT - Isolation and propagation: On specific hosts (ECE, TC and Lab. Animals). - Detection of virus nucleic acids: PCR, Hybridization, RFLP and Finger printing. - **Indirect Diagnosis** (Detection of virus specific antibodies) - Seroconversion Samples: Two serum one in the acute stage of the disease and the other in the convalescent stage in between 14 days. A suitable serological test is carried out on the two samples. If antibodies increased 4X is considered infection except in case of NDV and RP 2X only is considered infection. ## Sampling and Sample Preparation ### Objectives - Identify the basic measures that should be followed for ideal sampling. - Conclude the suitable samples for different viruses. - Practically prepare a soft tissue sample to be used in virus isolation. ### Characteristics of Ideal Virology Samples Specimens collected for isolation of viruses must have certain criteria like: - Aseptically as possible (of minimum contamination). - As fresh as possible (living animals or freshly dead animals within 2 hours after death). - From the anatomical sites of viral tropism. - Representative sample (one sample from each 1000 animal; 5 samples at least). ### General Considerations During Sampling - The nature of clinical sings: (Respiratory, intestine, nervous.....etc) - Respiratory sings in poultry (suspect: Newcastle disease virus "NDV", Infectious bronchitis virus "IBV", Infectious laryngotracheitis virus "ILT", avian influenza "AI".....). - Diarrhea in cattle (suspect: Bovine Rotavirus, Bovine Coronavirus...). - Age of herd or flock: - Each virus has a susceptible age as: Viruses causing tumor in poultry (Marek's disease virus "MDV" infects the poultry-at-any age and Avian leucosis virus "ALV" infects the poultry over 14 weeks of age). - Morbidity and mortality rate: - Viruses have different constant patterns of morbidity and mortality. - Morbidity rate = No. of infected animals / total No. of susceptible animals. - Mortality rate = No. of dead animals / total No. of susceptible animals. - Viruses causing respiratory sings in poultry: A) NDV and Avian influenza (high morbidity and high mortality). - B) IBV and ILT (moderate to low morbidity and mortality). - History of vaccination: Proper vaccination limits the chance of infection with the vaccinated viruses. - History of new entries: The appearance of a new viral disease or new viral strain may be sometime related to entrance of new animals to the flock or to the country. - The entrance of Foot and Moth disease virus "FMD" A serotype to Egypt through the importation live animals from Africa. - Season: The spread of some viruses are sometime related to season. - Arthropod-born viruses (ARBO): spread in summer season. - Influenza viruses: spread in winter season. - Geographical distribution: Some viruses are endemic in a geographical region and not in other(s). - Rinderpest virus "RP" (Absent-in-US-and-endemic in Africa and Asia). - Rabies virus (absent in UK). ### Sample Collection The sampling requires proper selection of both animals and organs. - **Selection of Animals** - Live animals (actual infected, apparently health or convalescent). - Dead animals (freshly dead "within 2 hours after death"). - **Selection of Organs** Proper sample must be collected from the anatomical sites of the viral tropism. ### Viral Tropism It is the affinity "ability" of certain virus to invade and replicate in a specific host tissues or cells. ### Factors Affecting The Viral Tropism - Presence of specific cellular receptors. - Temperature: MDV can replicate in feather follicles due to it need low temp. - pH: Rotavirus and Coronavirus can be replicate in alkaline pH "intestine" while the enteroviruses can be replicate in acidic pH "stomach". - Presence of certain metabolites: Rabies virus can be replicate in nervous tissues due to presence of catecolamines substance. ## Classification Of Viruses According To Tropism - Dermotropic: Pox, Herpes, Papplioma. - Viscerotropic: Rota, Corona, Entero - Neurotropic: Rabies, Avian encephalomyelitis, Equine encephalomyelitis. - Pneumotropic: Influenza, IBV, ILT. - Lymphotropic: Avian and bovine leucosis, MDV, Gumboro Virus. - Endotheliotropic: Bluetongue, African horse sickness. - Pantropic: NDV, RPV. ## Sample Transportation The specimen(s) should be collected in sterile, properly labeled vessels and sent to the laboratory with a complete history (type of sample, date, animal data "age, sex, number..." and suspected diagnosis). Also, the specimen(s) that collected by swaps should be dipped in transport medium (phosphate buffered saline "PBS", minimal essential medium "MEM" Hank's balanced salt solution "HBSS" and equal volume of glycerol in PBS "not used in RP sample"). The specimen(s) must be transported to the laboratory in ice box if the ambient temperatures are moderate and transit time to the laboratory is less than 1 day or in dry ice (-70°C) if the environmental temperature is high and transit times longer than a day. ## Sample Preservation The storage temperature of specimens depends on the properties of viral agent to be tested and duration of preservation. - Refrigerator (4°C): used for sample preservation up to 24 hours. If the viral agent to be isolated can be inactivated during this period, a portion of the specimen should be frozen at-70°C or lower. - Deep freezer (-20 and -40°C): used for sample preservation for weeks to few months. - Deep freezer (-70°C): used for sample preservation for months to few years. - Liquid nitrogen (-196°C): used for sample preservation for several years. - Lyophilization (Freeze-drying): (For long-term storage of virus suspensions). Dehydration of freeze viral suspension under high pressure and low temperature using a lyophilizer. The lyophilized material can be stored at 4°C. ## Types of Samples ### Types of Samples - **Viscous** - Sputum - Nasal discharge - Vaginal discharge - Semen - Saliva - Diarrhea - Cerebrospinal fluid - **Fluid** - Urine - Milk - Vesicular fluid - **Blood** - Whole blood - Serum - **Soft Tissue** - Liver - Spleen - Hart - Intestine - Brain - Kidney - Stool - **Hard Tissue** - Trachea - Joint capsule - Tendon ## Processing Of Samples ### Processing of Fluid Samples - Clarify the sample by centrifugation at 3000 rpm/30minutes. - Collect the supernatant. - Add antimicrobial agent to supernatant and allow standing for 1 hour at room temp. - Antibiotic as gentamycin (40 µg/ml). - Antimycoplasma as lincospectin - Antimycotic as Fungizone (2.5 µg/ml). ### Processing of Viscous-samples - Dilute the sample with PBS. - Complete as steps 1, 2 and 3 in fluid sample. ### Processing of Blood Samples - **Whole Blood** - Collect the blood with anticoagulant. - Centrifugation at 1500rpm/7-10 minutes. - Collect the different blood fractions separately. - **Serum** - Collect the blood without anticoagulant. - Leave the tube in an oblique position at 37°C for 1 hour. - Transfer the tube to refrigerator and leave it overnight. - Centrifugation at 5000rpm/10 minutes. ### Processing of Soft Tissue Samples - Grind the sample into small pieces using forceps and scissor. - Add equal amount of sterile sand. - Grind the sample with pistil till complete homogenization. - Dilute the sample 1/5 or 1/10 with PBS. - Collect the tissue homogenate in a centrifuge tube. - Apply 3 cycles of freezing and thawing for rupture of cells and release of virus outside the cells. - Clarify the sample by centrifugation at 3000 rpm/30 minutes - Collect the supernatant. - Add antimicrobial agent to supernatant and allow standing for 1 hour at room temp. ### Processing of Hard Tissue Samples - Homogenization of hard tissue using tissue homogenizer. - Complete as soft tissue sample from step 4. ## Questions - What is the purpose of freezing and thawing during the processing of virological samples? - What is the purpose for adding antimicrobial agents during sample preparation? - Why the degenerated samples not used in virus isolation? - Why RP suspected samples does not transported in (1:1) glycerol : PBS medium? ## Virus Isolation Virus isolation remains the gold standard for diagnosis of viral infections because it is the most sensitive available method developed until now. Theoretically, if a single virus particle is present in the specimen, it has the ability to replicate in the suitable host system and produce a characteristic lesion. Besides, virus isolation is the only method that provides a continuous supply of the virus strain for further identification, typing and vaccine or antigen production. Virus isolation requires at least a week, usually longer, expensive, needs a well-trained personal and a wide range of laboratory facilities. Virus can be grown from the suitably prepared specimen by inoculation into cell cultures, laboratory animals, embryonated chicken eggs, or the species of host animal from which the specimen was obtained. By far, the most widely used substrate is cultured cells. ## Virus Cultivation in Embryonated Chicken Eggs ### Introduction Embryonated Eggs were the most important host for virus isolation prior to the development of cell culture technique in the late 1940s. Now they are considered to be the most suitable host for isolation of many avian and some human viruses as well as for vaccine production. ### Objectives - Identify the suitable routes for inoculation of different viruses. - Fully describe the different steps of egg inoculation via all routes. - List the main requirements for development of an egg inoculation unit. ### Uses - Isolation of different viruses (many avian and some human viruses). - Characterization and identification of viruses. - Propagation of viruses for antigen and vaccine production. - Host for serum neutralization test. ### Advantages - Cheap (price, equipment and facilities). - Easily handle. - Easily marked. - Support growth of many viruses. - No immune system. ### Disadvantages Utilization of commercial eggs in virus inoculation is not recommended due to: - Some eggs may contain specific maternally-derived antibodies, which can interfere with virus growth during isolation or give misleading titration results. - Some eggs may carry latent infections (vertically-transmitted infection), which interfere with the virus growth and titration. Vaccines prepared from such eggs become a serious source of virus spread. - Examples of viruses cause latent infections: Avian leucosis. Avian encephalomyelitis. For the above reasons, specific pathogen-free eggs (SPF) are now used for virus isolation and vaccine production. ### Anatomy of the Egg - Amnion - Albumin - Yolk sac - Shell - Chorialantoic membrane - Developing embryo - Air sac - Shell membrane ### Routes of Inoculating Embryonated Chicken Eggs - **Route Of Inoculation** - Yolk sac - Amniotic cavity - Allantoic cavity - Chorioallantoic membrane (CAM) - **Age of Embryos** - 5-7 days - 7-11 days - 9-11 days - 10-12 days ### Steps of Inoculation in ECE - Candling: Viability, Age - Marking (Pencil) at specific sites - Yolk - Amniotic - Allantoic - Chorioallantoic membrane (Triangular area devoid of blood vessels and top of the air sac). - Surface sterilization of the inoculating site by 70% ethanol. - Poring of the shell at the indicated sites. - Yolk - Amniotic - Allantoic - Chorioallantoic membrane (Two pores one in the top of the air sac and other in triangular area devoid of blood vessels) - Inoculation of the virus suspension or suspected material (0.1-0.2ml) egg. - Sealing of pores. Medical plaster-Wax. - Incubation at 37°C and 40-60% humidity with daily observation. - First 24 hours deaths are considered non-specific except in case of velogenic viscerotropic New castle disease virus (VVNDV). - Period of incubation according to the type of virus. - Collection of viruses from different routes. - Chill the eggs overnight to kill embryos (prevent haemorrhage). - Collect the different component of eggs. ### Routes of Inoculation in ECE - **A** - Amniotic Inoculation 9-Day-Old Chick Embryo - **B** - Allantoic Inoculation 9-Day-Old Chick Embryo - **C** - Yolk Sac Inoculation 6-Day-Old Chick Embryo - **D** - Chorioallantoic Membrane Inoculation 12-Day-Old Chick Embryo ## Example of the Viruses Inoculating in ECE | Route | Age | Viruses | I/P | Harvest | Detection | Identification | |---|---|---|---|---|---|---| | Intra yolk sac | 5-7 days | Marek's disease virus. Avian encephalomyelitis virus African horse sickness virus. Blue tongue virus. | 5-7 | CAM | Pock lesions | Serological tests - AGPT - ELISA - FAT - SNT | | Intra Amniotic | 7-11 days | Primary isolation of Avian influenza Toga virus | 5-7 | Amniotic fluid | Rapid (Slide) Haemagglutination (HA) | Serological tests - HI - AGPT, ELISA - FAT,SNT | | Intra Allantoic | 9-11 days | New castle disease virus Influenza virus Egg drop syndrome virus Infectious bronchitis virus Herpes viruses | 5-7 | Allantoic Fluid | Rapid (Slide) Haemagglutination (HA) | HI, AGPT, ELISA, FAT,SNT | | On Chorioallantoic Membrane | 10-12 days | ILT-MDV (Herpes Viruses.) Fowl and cowpox-LSD (Pox Viruses) Avian Reovirus (Avian viral arthritis) | 5-7 | Choriallantoic Membrane (CAM) | Large coalescent pock lesions Pin headed pock lesions Pin pointed numerous pock lesions | Serological tests - AGPT - ELISA - FAT - SNT | - I/P: Incubation period. - TH: Till hatching. - ELISA: Enzyme linked immuno-sorbent assay. - ILT: Infectious larygeotracheitis - FAT: Fluorescent antibody technique. - MDV: Marek's disease virus - AGPT: agar gel precipitation test - SNT: Serum neutralization test. - HI: Haemagglutination inhibition test. - LSD: lumpy skin disease ## Pock lesions: - They are circumscribed, rounded, elevated foci developed on the surface of chorio-allantoic membranes as a result of virus multiplication. - Pock lesions may vary in size, color and distribution according to the causative virus. - Examples: 1- Large coasclent pock lesions (Caused by Marek's disease virus) 2- Pin headed pock lesions (Caused by Fowl poxvirus) ## Curling and dwarfing embryos: - (Caused by Infectious Bronchitis virus) ## Questions - Mention the importance of Embryonated chicken eggs? - How to isolate and identified infectious bronchitis virus on ECE? - Mention the causes of nonspecific deaths of inoculated ECE? ## Virus Cultivation In Cell Culture The technique of cultivating cells on a solid surface using a fluid overlay was originated and developed in the late 1940's. Until that time, laboratory animals and ECEs were the common systems used for virus isolation and propagation. In the next years, cell cultures from a wide variety of animal and human tissues were prepared and extensively used; as a result, most of the common viruses known today were discovered. ### Uses of Cell Culture in Virology - Primary isolation of different animal and human viruses. - Characterization and identification of different viruses. - Propagation of viruses for antigen and vaccine production. - Titration of different viruses either by infectivity titration or plaque assay. - Application of several serological tests (e.g. serum neutralization test, cell ELISA, immunofluoresnce and immuno-peroxidase). - Testing the antiviral activity of various chemicals and drugs. - Studying the growth curve and multiplication cycle of different viruses. - Preparation of monoclonal antibodies to a wide diversity of viral proteins. - Invitro expression of viral proteins. ### Advantages of Cell Culture - Support growth of most viruses. - Has no immune system to interfere with the virus growth. - Economic (high benefit with a considerable expense). - Low level of extraneous proteins that makes virus purification exhaustive. ### Disadvantages of Cell Culture - Latent infection especially in primary cell culture. - Variable sensitivity to different viruses. - Costs for establishing a tissue culture laboratory. ## Tissue Culture Laboratory Facilities ### Introduction Construction of a tissue culture laboratory or modifying an existing laboratory to accommodate tissue culture work is an essential preliminary task to the virologist before introducing the tissue culture techniques to his practice. In this section, the different materials needed for tissue culture are discussed briefly. ### Objectives - List the main requirements of a tissue culture laboratory. - Identify the suitable cases for using different laboratory facilities. - Differentiate between the different types of cells. ### Equipments - **Sterilization Equipment:** Sterilization of all materials used in tissue culture is the basic technical demand that would spoil all the practical procedures if not satisfactory. The nature of sterilization practice and equipment is basically dependent on the type of material to be sterilized either plastic, rubber, metal, glass or even fluid. - **Hot Air Oven:** Used for sterilization of metal instruments (e.g. scissors, forceps) and glass wares (e.g. bottles, pipettes, Petri dishes and roux). Complete sterilization is achieved after 2 hours at 200°C. - **Autoclave:** Used for sterilization of the different tissue culture materials (plastic, rubber, metal and glass) that can withstand high temperature (121°C) and high pressure. Fluids and buffers that do not contain any pertinacious material can also be sterilized by autoclaving. Complete sterilization by autoclaving is achieved after 20-30 minutes. - **UV Chamber:** Used for sterilization of tissue culture materials that can not withstand the high temperature and/or pressure (some plastic and rubber materials). Complete sterilization is achieved after 2 hours of ultraviolet exposure. - **Filtration System:** Used for sterilization of all fluids and reagents that contain proteinous material or components damaged by heat (eg. MEM, Serum, Trypsin-versin, antibiotic solution,....) This system utilizes a membrane filter with an average pore diameter of 0.22 µm that can trap a wide range of the contaminating macho organisms. - **Laminar Air Flow Cabinet (Workstation):** A continuous current of clean and sterile air is drawn over the working area through a HEPA filter (average pore diameter of 0.22 µm). This air current replaces the existing-contaminated environment and maintains sterile conditions - **Types** - **Horizontal Laminar Airflow Cabinet:** The air flow direction is horizontal. The HEPA filer is located in the back skeleton of the cabinet. Needs no glass cover in the working site. Suitable for culturing cells only. Virus inoculation in such cabinets is strongly not recommended. - **Vertical Laminar Airflow Cabinet:** The air flow direction is Vertical. The HEPA filter is located in the upper skeleton of the cabinet. Needs a glass barrier that covers two-thirds of the working site. Suitable for culturing cells and virus-inoculation. - **Importance:** Provides a complete sterile working space to avoid contamination of tissue culture media and cells. Protects the laboratory worker from being infected. Avoids dissemination of highly contagious pathogens to the nature. - **Inverted Microscope:** It is an ordinary light microscope with the light source up and the lens system down. Used for examination of the tissue culture cells and identification of the virus-induced cytopathological changes. - **Incubator:** Used for maintenance of the tissue culture cells in suitable environmental conditions that support its viability and growth. Incubators may be: ordinary (promotes suitable temperature) or CO2 (promotes suitable temperature and CO2 tension). - **Automatic Pipetting Aid:** Used for sterile pipetting (suction and pumping) of fluids and reagents during tissue culture practice. ### Instruments and Consumables - Glass pipettes (1, 2, 5, 10, 20 ml). - Petri dishes of different diameters. - Bottles of different sizes. - Tubes of different forms and capacities. - Scissors and forceps. - Flasks and cylinders (different sizes). - Funnels. - Cryovials (for preservation of cultured cells). - Cell scrapers. - Syringe filters of 0.22 µm and 0.45 µm pore diameter. - Pipette tips of different capacities. ### Buffers and reagents - Phosphate Buffered Saline (PBS). - Trypsin solution (0.25%). - EDTA solution (0.1%). - Trypsin-Versin solution (Trysin 0.25% and EDTA 0.1%). - Antimicrobial solutions (e.g. antibiotics, antimycotics). - Dimethyl sulfoxid (DMSO). - Trypan blue (0.4%). - Ethanol (70%). ### Vessels Tissue culture vessels are sterile, transparent containers that are used for maintenance and growth of cultured cells. Many forms of the vessels are now available to fit different users' requirements and variable experimental designs. The tissue culture vessels are classified according to the material from which they are synthesized into: - **Glass (Resterilizable) Vessels** - Small and large sized glass bottles (roux). - Roller bottles. - **Plastic (Disposable) Vessels:** (Tissue culture prescriptions): - Small size Surface area 25 cm³). - Medium si Face area 75 cm³). - Large size Ace area 150 cm³). - **Tissue Culture Plates:** (6, 24, 48 and 96 wells) - **Plastic Petri dishes** of different diameters (surface area). - **Cell Culture Tubes** They can also be classified according to their system of ventilation into: - **Open System:** A continuous gas exchange between the internal vessel environment and outer incubator environment. Requires CO₂ incubator to avoid loss of CO2 from the culture vessel, which leads to gradual alkalinity in the culture media and further death of cells. e.g. Tissue culture plates, ventilated flasks, Petri dishes. - **Closed System:** There is no gas exchange between the internal vessel environment and outer incubator environment. Can be incubated in any incubator (ordinary or CO2). e.g. Tissue culture bes, tightly-closed flasks, Roux, roller bottles. ### Medium Cells have complex nutritional requirements that must be met to permit their propagation in vitro (carbohydrates, amino acids, minerals, vitamins, growth factors,....e Different types of cells have differential growth requirements and a number of chemically-defined formulations have been developed to support the growth of a wide variety of cell lines (e.g. MEM, RPMI, DMEM, M199,...etc). ### Media Additives - **Serum:** Promotes cell growth and multiplication. It should be heat-inactivated at 56°C for 30 minutes to inactivate complement prior to its addition to-culture-media. e.g. Fetal calf serum - newborn calf serum - horse serum. - **Sodium bicarbonate:** Used as a nutrient and a buffering agent to adjust the pH of medium. - **Antibiotics:** To overcome the accidental bacterial contamination of culture. e.g. Penicillin and streptomycin - Gentamycin. - **Lactalbumin hydrolysate:** (promotes cell growth and multiplication) - **Phenol red:** (pH indicator). - **Special requirements:** (needed for certain types of cells) e.g. Non-essential amino acids, insulin, purvate and yeast extract. ### Balanced Salt Solution - Normal saline or PBS. - Sufficient to maintain cell viability for few hours without providing the minimal growth conditions. ### Maintenance Medium - Cell culture medium containing 1-2% Serum. - Sufficient to maintain cell viability for 2-3 weeks without providing optimum conditions for cell division and growth. ### Growth Medium - Cell culture medium containing 5-10%. - This medium provides optimum conditions for cell division. ## Cells Tissue culture cells are originated from different sources of normal and abnormal cells from many species. They are generally separated into three types according to their origin: - **Primary Cell Culture** prepared directly from plant, animal or human tissues and lives in culture for a short period (2-4 weeks). - e.g. Chicken embryo fibroblast (CEF); chicken embryo liver (CELi) and Fetal bovine kidney (FBK). - **Diploid Cell Strain** usually derived from primary cell culture and continued to grow in culture for longer periods (up to 1 year). - e.g. Human embryonic lung, human newborn foreskin and WI-38. - **Established Cell Line** prepared directly from tumor cells or by carcinogenesis of normal cells using one of different means (e.g. chemically, by radiation or using certain tumor viruses). - e.g. Madin Durbey bovine kidney (MDBK); African green monkey kidney (Vero) and Baby hamster kidney (BHK). ### The Differential Criteria of These Cell Types | Item of differentiation | Primary Cell culture | Diploid Cell strain | Established Cell line | |---|---|---|---| | Type of Cells | Normal | Normal | Abnormal (tumor) | | Cell structure and function | Maintained | Maintained | Lost | | Chromosomal number | Diploid (2n) | Diploid (2n) | Heteroploid (>2n) | | Number of subcultures | 1 or 2 | 20-50 | Unlimited | | Contact inhibition phenomenon | Present | Present | Absent | | Over growth | No | No | Yes | ### Anchorage Phenomenon - **Definition:** It is the ability of cells to attach firmly with the culture vessel surface and form a monolayer sheet of cells. - **Cell Types** - **Adherent (anchord) cells:** Cells that possess the anchorage phenomenon. Mainly epitheloid and fibroblast cells. e.g.: MDBK, Vero, BHK, - **Suspended (non-adherent) (non-anchord) cells:** Cells that lack the anchorage phenomenon. Remains suspended in the culture media. Mainly derived from blood cells. e.g.: Myeloma, SF-9, High five,... - **N.B.** Adherent cells may be suspended if grown in stirred vessels for intensive production of vaccines and antigens. ### Factors Affecting Anchorage Phenomenon - Type of cells: (anchored or suspended). - Viability of cells: (only viable cells have the anchorage phenomena). - Incubation temperature: (most cells anchored at 37°C). - Culture vessel surface: (It should be positively charged). - Media components: (Presence of Calcium ions and fibrinonectin is essential for anchorage phenomenon). ## Common Tissue Culture Techniques ### Preparation of Primary Cell Culture "Chicken Embryo Fibroblast (CEF)" ### Introduction Primary cell cultures are obtained directly from animal's tissues and organs. These cells have a short life span in the laboratory since the culture dies after a few passages. They usually contain a variety of cell types and consequently support growth of a wide variety of viruses. Primary cultures may be obtained from embryos or adult animals. Embryonic cells are rapidly dividing, can grow easily in vitro and free from the microflora contamination commonly observed with adult animal's cells. In the following section, preparation of primary cell culture from the chicken embryo fibroblast cells will be described. ### Objectives - Illustrate the main steps for preparation of primary tissue culture. - Predict the reasons for getting poor primary cell culture. - Demonstrate how to apply the same technique for preparation of primary tissue culture from a bovine kidney. ### Materials - Magnetic Stirrer. - Manual or automatic pipetting device. - Bench-top centrifuge. - Incubator (ordinary or CO₂) - Haemocytometer. - Petri dishes (about five). - Scissors and forceps. - Side-armed flash (500 ml capacity). - Receiving flask (500 ml capacity). - Funnel covered with double layer of gauze. - Centrifuge tubes (15 ml capacity). - Culture vessels (suitable type and number). - ECE (8-11 days-old). - Growth media (MEM + 5-10% FCS). - Trypsin 0.25%. - Fetal calf serum (FCS). - MEM + 3X Antibiotic solution. ### Procedure - Candle the egg to determine presence, viability and age of the embryo. - Swab the egg shell by 70% ethanol. - Carefully remove the shell covering the air sac and reflect the shell membrane. - Pick-up the embryo by a sterile forceps without piercing the yolk sac. - Transfer the embryo to a petri dish and remove appendages (head, wings and legs) and viscera using a sharp scissors. - Transfer flesh into another petri dish containing MEM and antibiotic. - Wash 3X in different disches. - Transfer into a petri dish and cut into fine pieces using sharp scissors. - Place the minced flesh into a sterile side-armed flask with a magnetic bar and cover with trypsin solution (0.25%), then stir for 3-5 minutes. This step is termed Trypsinization and aimed to separate individual cells. - Pour off the trypsinzation supernatant into a receiving flask containing fetal-calf serum through_a_funnel covered with_double layer of gauze to hold the cell-clumps. This step is termed Antitrypsinization and aimed to inactivate the trypsin to avoid its extensive action on cells. - Repeat the trypsinization and Antitrypsinization steps for 3-5 times. - Split the collected suspension into sterile centrifuge tubes and pellet at 1500 rpm for 5-10 minutes. - Decant the supernatant and resuspend the pellet in 5 ml growth medium. - Count the cells using a haemocytometer. - The cells should be count in the large squares located at peripheries of the haemocytometer (each contains 16 small square). - Cells are counted in the small squares in L shape form (cells located internally, in the right and lower borders are only counted). - Extension of counting from square to another occurs in zigzag manner - Only viable cells should be counted (preferred to stain with Trypan blue) - Dilution factor for dispensing cells in culture vessel is calculated by the following formula: - Number of cells in the large square X 10¹ - Desired number of cells / ml - Resuspend cells in growth medium according to the dilution factor and dispense into suitable culture vessels. - Incubate till cells reach the desired confluency. ### Questions - Why do we need to remove appendages and viscera from embryoes used for preparation of primary chicken embryo fibroblast? - Why do we need to count cells before dispensing into culture vessels? - What is the purpose of repeating trypsinization and anti-trypsinization steps? - Why we do not allow trypsinization for 15 min? - What is the function of serum used in the receiving flask? - In preparation of primary bovine kidney cells, identify the following steps: ## Subculture Technique ### Introduction Tissue culture cells are attached to the vessel surface via protein receptors, flattened and grown to yield a continuous sheet. This sheet is usually one cell thick and is called monolayer. When the monolayer becomes confluent (covers the entire surface of the vessel), some cells should be transferred to another vessels to allow further expansion of cells (subculture). ### Objectives - Describe the main steps for subculturing invitro cultivated cells. - Recognize importance of the sterile technique in cell culture laboratory. - Specify the conditions require subculturing of tissue culture cells. ### Aims of Subculture: - Expansion of tissue culture cells to maintain a continuous source. - Avoiding aging and overgrowth of cells. - Preparation of host cells for virus inoculation and titration as well as for many serological tests like SNT, cell ELISA and FAT. ### Materials - Laminar Air Flow (Horizontal or vertical). - Incubator (ordinary or CO₂). - Manual or automatic pipetting device. - Haemocytometer and coverslips. - Tissue culture flask(s) containing confluent monolayer(s) of cells. - Culture vessels (suitable type and number). - Pre-warmed growth media (MEM + 5-10% FCS + antibiotic). - Pre-warmed Trypsin-Versin solution (0.25% Trypsin + 0.1% EDTA). - Fetal calf serum (FCS). - Phosphate buffered saline (PBS). - Sterile glass pipettes of different sizes. ### Procedure: - Discard (pour