Aseptic Techniques

10 Key excerpts on "Aseptic Techniques"

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  eBook - PDF

  Freshney's Culture of Animal Cells

  A Manual of Basic Technique and Specialized Applications

  • Amanda Capes-Davis, R. Ian Freshney(Authors)
  • 2021(Publication Date)
  • Wiley-Blackwell

    (Publisher)

  CHAPTER 12 Aseptic Technique Aseptic technique refers to a set of practices that are used to maintain asepsis, or the absence of microorganisms. Origi-nally developed by Louis Pasteur to handle microbial cultures, aseptic technique was adapted by Robert Koch to control infection following surgical procedures (Schlich 2012). Alexis Carrel and other early tissue culture pioneers were practition-ers of aseptic technique and invented flasks and instruments that allowed aseptic handling of cultures (Carrel 1923). However, many of the early techniques were considered overelaborate and slowed down handling of cultures. A better understanding of tissue culture has enabled us to identify the key elements of an aseptic environment and to develop a simple set of handling practices. The resulting procedures act as a foundation upon which all other tissue culture techniques are based. 12.1 OBJECTIVES OF ASEPTIC TECHNIQUE 12.1.1 Managing Contamination Risk Contamination is a major problem in tissue culture and a constant threat to good cell culture practice (GCCP) (see Section 7.2.1). Bacteria, yeast, fungal spores, and viruses may be introduced via tissue culture personnel, the atmosphere, work surfaces, reagents, and other sources (see Section 16.1). Cells from other cultures may also be introduced, resulting in cross-contamination (see Section 17.2.2). Contamination can be minor and confined to one or two cultures, can spread among several cultures and compromise a whole experiment, or can be widespread and wipe out your (or even the whole laboratory’s) entire stock of cells. Freshney’s Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications , Eighth Edition. Amanda Capes-Davis and R. Ian Freshney. © 2021 John Wiley & Sons Ltd.

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  Upstream Industrial Biotechnology, 2 Volume Set

  • Michael C. Flickinger(Author)
  • 2013(Publication Date)
  • Wiley

    (Publisher)

  Chapter 4: Cell Culture, Aseptic Techniques John M. Davis

  University of Hertfordshire, Hatfield, School of Life Sciences, Hertfordshire, United Kingdom

  Kevin L. Shade

  Novartis Vaccines and Diagnostics, Speke, Liverpool, United Kingdom

  4.1 Introduction

  4.1.1 Microbial Contamination

  We live in a world in which we are constantly surrounded by microbes, in our environment as well as upon and within our own bodies. Yet the successful performance of cell culture demands that we be able to maintain and manipulate our cultures free of all microbial contamination. As we cannot sterilize our cultures after manipulating them, we are completely dependent on Aseptic Technique to ensure that, in handling our reagents, cells, and all the associated equipment, we maintain the sterile environment that our cultures require.

  Aseptic technique is a combination of many procedures all designed with the single goal of minimizing the probability of a microbe gaining access to the cell culture environment. It is important to appreciate this concept of minimizing the probability of contamination, as even the best aseptic technique cannot absolutely guarantee the maintenance of sterility (indeed all sterilizing techniques work on this same principle (1)). Thus, a methodical and fastidious approach is required, with attention to each element of each procedure. Dropping an element or cutting corners may not necessarily result in an actual contamination the first time, but will increase the probability of a contamination occurring. Consequently, it is essential that all the elements of an aseptic procedure are carried out consistently every time, making sterility breakdowns rare events.

  4.1.2 Cellular Cross-Contamination

  Cell culture requires more however, than just the type of aseptic technique used for the exclusion of microbes. Our work becomes at best meaningless, and at most potentially dangerous if we are not culturing the cells we think we are. Thus aseptic technique for cell culture must include procedures to minimize the possibility of contaminating one cell line with another. Evidence that such inadvertent cross-contamination could occur was presented in the 1970s when various cell lines from a number of different laboratories were all shown by isoenzyme analysis and/or karyotyping to be, in fact, HeLa cells; these findings were later extended to include contamination involving cells other than HeLa (2–4). Thus the second aim of all aseptic technique used for cell culture is to minimize the probability of contaminating our pure characterized cultures with microbes or other cells.

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  Culture of Animal Cells

  A Manual of Basic Technique and Specialized Applications

  • R. Ian Freshney(Author)
  • 2015(Publication Date)
  • Wiley-Blackwell

    (Publisher)

  and Table 19.1). Aseptic technique aims to exclude contamination by establishing a strict code of practice and ensuring that everyone using the facility adheres to it.

  Contamination can be minor and confined to one or two cultures, can spread among several cultures and compromise a whole experiment, or can be widespread and wipe out your (or even the whole laboratory’s) entire stock. Catastrophes can be minimized if (1) cultures are checked carefully by eye and on a microscope, preferably by phase contrast, every time that they are handled; (2) cultures are maintained without antibiotics, preferably at all times but at least for part of the time, to reveal cryptic contaminations (see Section 12.4.8); (3) reagents are checked for sterility (by yourself or the supplier) before use; (4) bottles of media or other reagents are not shared with other people or used for different cell lines; and (5) the standard of sterile technique is kept high at all times.

  Mycoplasmal infection, invisible under regular microscopy, presents one of the major threats. Undetected, it can spread to other cultures around the laboratory. It is therefore essential to back up visual checks with a mycoplasma test, particularly if cell growth appears abnormal (see Section 14.3.2).

  5.1.2 Maintaining Sterility

  Correct aseptic technique should provide a barrier between microorganisms in the environment outside the culture and the pure, uncontaminated culture within its flask or dish. Hence, all materials that will come into direct contact with the culture must be sterile and manipulations must be designed such that there is no direct link between the culture and its nonsterile surroundings. It is recognized that this sterility barrier cannot be absolute without working under conditions that would severely hamper most routine manipulations. As testing the need for individual precautions would be time consuming, procedures are adopted largely on the basis of common sense and experience. Aseptic technique is a combination of procedures designed to reduce the probability of infection, and the correlation between the omission of a step and subsequent contamination is not always absolute. The operator may abandon several precautions before the probability rises sufficiently that a contamination is likely to occur (Fig. 5.1

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  Sensors in Bioprocess Control

  • John Twork(Author)
  • 2020(Publication Date)
  • CRC Press

    (Publisher)

  The pharmaceutical industry manufacture of human drugs or parenterals requires effective sterilization techniques using chemicals, saturated steam, dry heat, irradiation, and other methods for eliminating unwanted microorganisms. These sterilization methods have been applied in the rapidly growing biological process industries. The requirement for aseptic conditions in biological reactions, aseptic media fills, and final parenteral drug packaging is now regulated by agencies such as the Food and Drug Administration (FDA). The use of current good manufacturing practices (CGMPs) and good laboratory practices is designed to apply proper methods of sterilization for specific biological processes.

  _____________* Present affiliation: Lear Siegler Measurement Controls Corporation, Englewood, Colorado

  At the present time, the art of aseptic sampling in biotechnical facilities is expanding, with equipment manufacturers offering manual and automatic sampling devices for installation in the biological fermentation equipment, bioreactors, tanks, and associated equipment. Standard procedures for performing aseptic sampling is primarily the responsibility of the user of the equipment, although some guidelines are provided by the equipment manufacturer and engineer/designer of the plant.

  However, a wide variation in methods for sampling these process equipment exists today, and more uniform methods and procedures are needed in the industry. The variety of processing and equipment requirements in this new industry complicates this goal as standardization efforts continue to grow. For example, the American Society for Testing and Materials (ASTM) has organized the E-48 Committee on Biotechnology during the past four years to address standard test methods and procedures for identification of microorganisms, terminology, and process control to containment, environmental, toxicity, and disposal issues. ASTM currently has a draft Standard Practice for Aseptic Sampling being developed for eventual use by the industry. Of the 5000 ASTM standards currently available, about 200 address sampling, but the E-48 committee is producing the first aseptic sampling standard practice for industrial application.

  B. Challenges in Proper Sampling

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  Introduction to Biotechnology and Biostatistics

  • Khushboo Chaudhary(Author)
  • 2019(Publication Date)
  • Delve Publishing

    (Publisher)

  Biotechnology Practical 13 13.1. STERILIZATION TECHNIQUES (EXPERIMENT 1) 1. Objective Performing different methods of sterilization used in biotechnology. 2. Principle Maintaining the sterile environment during the transfer or culturing of cell/ tissues of microbes plants and animal cells is most important. The concept of sterilization, for making the materials free from the any type of contamination was given by L. Pasteur. Thus sterilization is a process of making an article. surface or medium free from any type of microorganisms that contaminate the object and provide unwanted results. However, sterilization is one of the most important steps for cultivation isolation and study of purified cells or tissues in the laboratory. The other important things to be sterilized are the surgical tools, culture vessels, nutrient media, and plant materials. Some other methods used to make these sterile are disinfection and incineration. (a) Disinfection: Disinfection is a similar process of killing the harmful microorganisms especially the objects but not the culture media. Disinfection of table tops, equipment and others surface are usually done by using glycolic acid compounds, carbolic acid and formaldehyde, ethanol, etc. (b) Incineration: It is a process of killing of microorganisms by using flame, Introduction to Biotechnology and Biostatistics 252 therefore, it is called flame sterilization. It is done by keeping the inoculation needle over the flame of Bunsen burner till it become red hot. Thus, the microorganism present on the surface of needle are destroyed. Beside, physical and chemical methods are used for sterilization. I. Physical methods of sterilization There are several physical methods of sterilization of materials and objects. These are briefly described below: (a) Moist Heat: Culture media (liquid and agar), water, glassware, surgical blades and scalpels are sterilized by using moist heat, i.e., steam under pressure.

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  Microbiology and Chemistry for Environmental Scientists and Engineers

  • Jason Birkett, John Lester(Authors)
  • 2018(Publication Date)
  • CRC Press

    (Publisher)

  Although the general incidence of pathogens in the working environment may be quite low it should be remembered that an integral part of many laboratory techniques is the intentional promotion of optimum growth of organisms which is rapid and prolific compared to their normal growth in the natural environment. The slightest contamination can therefore lead to rapid and unintentional proliferation of pathogens unless certain precautions are adhered to.

  A mandatory precaution is the use of Aseptic Techniques at all times. This involves the use of apparatus and materials which are initially rendered free of living organisms and which, when in use, are manipulated in a manner which excludes the possibility of chance contamination from external sources.

  5.3 Aseptic Techniques

  5.3.1 Sterilization

  Sterilization is the process of removing or destroying all living organisms associated with a particular material. After treatment, provided that the material remains unexposed to external sources of contamination, sterilization should be effective permanently. Sterilization can be achieved by exposing the material in question to lethal physical or chemical agents or by the selective removal of micro-organisms from liquids by filtration.

  (a) Heat

  Heat is the most commonly used agent for sterilization. Laboratory glassware, pipettes, metal objects and other heat-resistant items can be sterilized in a temperature-controlled oven at 170°C for 2 h. As with all sterilization methods, ample provision for penetration of the lethal agent has to be made. If the oven is fairly well filled or if glassware is closely packed in metal containers, for example, a longer period, perhaps 3 h, should be used to ensure sufficient heating throughout. A hot air oven is obviously unsuitable for liquids and many heatlabile materials including plastics. Sterilization can be achieved by moist heat, which is effective at lower temperatures. Boiling of aqueous solutions will kill the majority of vegetative organisms present, but this is ineffective for bacterial endospores which can germinate and grow once the boiled medium has cooled. Heating to 100°C for 30 min on each of three successive days is necessary to kill bacterial spores which germinate in the periods between heating cycles.

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  Plant Cell Culture

  • Julian Coleman, David Evans, Anne Kearns(Authors)
  • 2020(Publication Date)
  • Taylor & Francis

    (Publisher)

  Chapter 5

  Culture facilities, sterile technique and media preparation

  1. Introduction

  Successful plant tissue culture requires laboratory facilities where aseptic conditions can be established and maintained. Aseptic technique is critical for plant tissue culture practice because the media and the environment in which plant materials are grown, are also ideal conditions for microorganisms to proliferate. Fungal and bacterial contamination are amongst the hardest to deal with, as these organisms rapidly outgrow plant material and change the defined conditions of the growth medium resulting in poor growth or death of the plant culture. These deleterious effects are brought about by the contaminating organism(s) consuming the culture nutrients, excreting metabolites into the growth medium or colonizing the plant tissues. The basic Aseptic Techniques of handling and culturing plant materials were developed over many years and are designed to ensure that no contaminating micro-organisms enter the culture. Although not always essential, a carefully designed tissue culture unit is recommended to preserve sterile conditions and to achieve consistent results.

  2. The basic laboratory layout and equipment

  The prime purpose of a tissue culture laboratory is to enable the processing and culture of plant material in a sterile environment. To facilitate this purpose, certain basic facilities are required. These usually include the following:

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  Bacteriology Methods for the Study of Infectious Diseases

  • Rowena Jenkins, Sarah Maddocks(Authors)
  • 2019(Publication Date)
  • Academic Press

    (Publisher)

  Chapter one

  Fundamental skills for infectious disease research

  Abstract

  This chapter takes you through all of the practical considerations you will need to address before you enter the bacteriology laboratory, as well as the basic skills you are likely to need once you have started your work. From setting up the initial laboratory bench space through to assessing the risk of the work to be carried out, this chapter allows the reader to master basics such as aseptic pipetting, media preparation and filtering of solutions. It will also provide details on the units and calculations you will come across and the equipment that you will use in your day-to-day laboratory experience.

  Keywords

  Aseptic technique; Bacteriology; Calculations; Media preparation; Microbiology; Pipette; Risk assessment; Sterile technique

  1. 1.1 Introduction
     1. 1.1.1 Health and safety
     2. 1.1.2 Basic laboratory preparation, kit and consumables
     3. 1.1.3 Pipettes

  1. 1.2 Aseptic technique
  2. 1.3 Common laboratory units
  3. 1.4 Top tips for working safely in the lab
  4. 1.5 Notes page

  1.1. Introduction

  When planning any experiment, it is important to make sure that all of the equipment and reagents are pre-prepared and arranged within a sensible working area so that disruption of your work is minimal. This includes considering whether ice is available for reagents that need to be cold and somewhere to dispose of tips, glassware, and waste solutions, for example. This chapter will help you to consider the risks that need to be assessed before you start and what you need to set up to carry out experiments you are planning. This information will be applicable irrespective of the type of experiment you do.

  1.1.1. Health and safety

  Before you can start in the laboratory, it is essential that you take some time to consider procedures that might need to be in place to allow you to work safely.

  When working within the microbiology laboratory, you are going to be handling microorganisms. It is important for you to know what kind of risk they might pose to you and anyone else working within the laboratory. Because the microbial cultures you are going to be handling can often contain many millions of potentially infectious cells, guidelines are in place to help you handle them safely. The Advisory Committee on Dangerous Pathogens provides an approved list of biological agents (microorganisms) as referred to in Control of Substances Hazardous to Health (COSHH), which includes bacteria, fungi and viruses that could pose a risk to human health. You should refer to this if you are uncertain about the hazard rating of the organism with which you are working. Broadly, the biological agents are split into four categories (Table 1.1

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  Scientific Basis Of Tissue Transplantation, The

  • Abdul Aziz Nather, Glyn O Phillips(Authors)
  • 2001(Publication Date)
  • World Scientific

    (Publisher)

  SECTION IV: STERILE TECHNIQUES This page is intentionally left blank Advances in Tissue Banking Vol. 5 © 2001 by World Scientific Publishing Co. Pte. Ltd. 15 PRINCIPLES OF STERILE TECHNIQUE S.Z. MORDIFFI Major Operating Theatre Suite Nursing Department, National University Hospital 5 Lower Kent Ridge Road, Singapore 119074 A. NATHER N U H Tissue Bank, National University Hospital 5 Lower Kent Ridge Road, Singapore 119074 1. Introduction All tissue-bank operators must be trained on aseptic technique in order to perform sterile procurement of tissues from living and deceased donors. It is vital that each new technologist be attached to the operating room for at least two or three months to learn and practise hands-on all principles of sterile technique. The technologist must learn not only how to scrub and gown in a sterile fashion. He or she must also learn how to maintain sterility in the operating room as well as the methods of sterilising equipment and materials in the theatre sterile supply unit. 2. Sterile Technique in the Operating Room Sterility is the "absence" of microorganisms including spores (Groah, 1990). Sterility is attained by the process of sterilisation. Aseptic 235 236 S,Z. Mordiffi & A. Nather technique is the procedure of ensuring that an item remains sterile after sterilisation and at the time of use for the patient. It is the responsibility of the surgical team to ensure that only sterile items are used during surgery (Atkinson and Fortunato, 1996). The skin is a barrier to microbial infection. A surgical incision is a potential port of entry for microorganisms (Groah, 1990). There- fore, items used during surgery must be sterile as non-sterile item carries with it pathogenic microorganisms which may cause wound infections. Aseptic technique is the method by which contamination of microorganisms is prevented.

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  Microbial Culture

  • Susan Isaac, Prof David Jennings(Authors)
  • 2020(Publication Date)
  • Garland Science

    (Publisher)

  4 Principles and Initial Steps in Culturing 4.1 Aseptic Techniques and manipulations The handling and culture of micro-organisms are carried out using Aseptic Techniques, the primary aims of which are to keep the test microbe in, and other microbes out of, the culture. This ensures that the microbial culture under test, the laboratory worker and the surroundings are kept free from contamination. At first, some of the operations may seem unwieldly and difficult to carry out but, as with other specialist techniques, practice helps to improve success rate. All apparatus and culture media must be sterilized prior to use, so that it is completely free from microbial contamination. Any equipment that comes into contact with microbes must also be sterilized after use and before disposal. It is most usual to sterilize laboratory equipment by heating with steam, under pressure (autoclaving) although a conventional pressure cooker can be used for small-scale sterilization. By heating at increased pressure, the temperature is raised above 100°C and therefore the time for which heating is required can be kept as short as possible. Autoclaving can affect the components of nutrient medium but the effect can be minimized (but not removed) by limiting the heating time. Normal autoclave conditions are 15 min at 121°C (15 lb in ‒2 ; 10 2 kPa). Temperature and pressure can be closely controlled. The sterilization chamber of the autoclave may be aligned vertically, most usual for large-capacity versions, or horizontally. During sterilization, solutions may boil vigorously and bubble within vessels. It is, therefore, advisable to place laboratory media and solutions for autoclaving in large containers, for example autoclave a 100 ml solution in a vessel with a 200–250 ml capacity. For autoclaving, screw caps of vessels should be placed on loosely and tightened after vessels have been removed from the autoclave, after sterilization

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