Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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public drainage system. It is therefore necessary to

neutralize or massively dilute any acid or alkaline

wastes before they can be disposed of by such a route.

0050 Similar dilution is also required before even small

quantities of water-miscible solvents such as acetone

or ethanol can be disposed of down the drain.

Accumulation of solvent vapors in sewers would

pose a considerable risk to maintenance workers

and, indeed, to the system itself. Consequently, only

very limited use can be made of the drains for disposal

of liquid waste, and it is necessary to ensure that any

waste so consigned does not contain significant

concentrations of heavy metals or other toxic

substances.

0051 In considering discharge to the local environment,

discharge of vapors and fumes from local exhaust

ventilation and fume cupboard outlets must not be

overlooked. Adequate dilution must occur before the

fumes are discharged, and, if possible, noxious mater-

ials should be trapped at source rather than simply

vented to the atmosphere.

0052 Most laboratory waste is now destined either for

high-temperature incineration or for burial at a duly

licensed landfill site, and the producer of the waste is

deemed to be responsible for it right up to the moment

of its ultimate disposal. This gives rise to the so-called

‘cradle to grave’ view of the producer’s responsibility.

0053 The onus upon the producer to ensure proper dis-

posal is, in any case, a heavy one. Suitable and separate

containers should be available in the laboratory for the

collection and segregation of various types of waste. In

particular, flammable solvents should be segregated

from nonflammables. Halogenated solvents should

be collected separately, as far as possible, and must

not be mixed with ketones. In general, care must be

taken not to bulk waste, especially solvent waste, in

such a manner as to create additional hazards by the

mixing of incompatible materials.

0054 Producers must ensure that laboratory waste is

clearly and adequately labeled as to its nature and

hazardous properties, securely packaged, and dis-

posed of via an authorized contractor. Moreover,

they should satisfy themselves as to the methods to

be employed by the disposer, and this may involve

visits to the treatment plant or landfill site. Some

types of waste may require separate secondary con-

tainment before packaging for transportation, espe-

cially highly reactive materials, oxidizing agents and

foul-smelling substances. Others may need to be seg-

regated simply because of a high toxicity hazard. This

certainly applies to carcinogens, and conspicuous

labeling is essential in such cases.

0055 Finally, detailed written records of all waste dis-

posals should be made and retained for at least two

years.

See also: Effluents from Food Processing: Disposal of

Waste Water; European Union: European Food Law

Harmonization; Quality Assurance and Quality Control

Further Reading

COSHH (1999) Control of Substances Hazardous to Health

(Approved Codes of Practice) – Control of Substances

Hazardous to Health Regulations 1999. London: HMSO.

Health and Safety Executive (1997) Successful Health and

Safety Management, 2nd edn. London: HSE Books.

National Institute for Occupational Health and Safety

(2001) Registry of Toxic Effects of Chemical Substances.

NIOSH: Cincinnati, OH.

Royal Society of Chemistry (1992) In: Luxon SG (ed.)

Hazards in the Chemical Laboratory, 5th edn. London:

Royal Society of Chemistry.

Royal Society of Chemistry (1989) Safe Practices in

Chemical Laboratories. London: Royal Society of

Chemistry.

Microbiological Safety

V N Scott and I Walls, National Food Processors

Association, Washington DC, USA

Introduction

0001Food laboratories may handle potentially infectious

materials, e.g., foods contaminated with pathogenic

microoganisms such as Salmonella spp., Escherichia

coli O157:H7, Listeria monocytogenes, Shigella,

and even Clostridium botulinum. Numerous pub-

lished reports have documented laboratory transmis-

sion of infectious agents, including those from E. coli

O157:H7, Salmonella, and Shigella, although infec-

tion occurs primarily in clinical laboratories. The

incidence of laboratory transmission of foodborne

pathogens is not known but is believed to be very

low. Nevertheless, the risk of exposure exists; this

risk can be reduced to a very low level by use of good

laboratory practices and containment techniques

(biosafety level 2), as described in this chapter.

Interestingly, laboratory-acquired infections with

Cryptosporidium parvum have been reported in

almost every laboratory working with this parasite;

therefore, it is important to adhere strictly to

all precautions to prevent transmission of this

agent. Good laboratory practices are particularly im-

portant when it is recognized that fewer than 20% of

LABORATORY MANAGEMENT/Microbiological Safety 3443

laboratory-associated infections are attributed to a

known accident.

0002 The most frequently recognized causes of labora-

tory infections are oral aspiration due to mouth pip-

etting, inoculation with syringes and needles, animal

bites, spray from syringes, and centrifuge accidents.

Other recognized causes include cuts from contamin-

ated glassware and the spilling of pathogenic cultures.

Exposure to infectious aerosols is considered to be the

most likely source for cases not associated with a

known accident.

0003 Biosafety practices relating to sample handling,

containment of biological hazards, disinfection and

decontamination in the laboratory and personnel

practices will be covered in this article. A list summar-

izing microbiological safety practices can be found at

the end. These guidelines should be considered

minimal guidelines. They should be customized for-

individual laboratories after consulting additional

references.

Biosafety Levels

0004 There are four biosafety levels for working with

microorganisms in the laboratory. These consist of a

combination of laboratory practices and techniques,

safety equipment, and laboratory facilities that pro-

vide increased protection to personnel, the environ-

ment, and the community as the level increases.

Biosafety level 1 is suitable for work involving well-

characterized agents not known to cause illness con-

sistently in healthy persons; work is generally

conducted on open benchtops using standard micro-

biological practices. Biosafety level 2 is suitable for

work involving agents of moderate potential hazard

to personnel and the environment. The primary

differences between biosafety level 1 and 2 is that,

with the latter, extreme precautions are taken with

contaminated sharp items and certain procedures

that may create aerosols are conducted in biological

safety cabinets or other physical containment equip-

ment. Biosafety level 3 is applicable to laboratories

working with agents that may cause serious disease

via the inhalation route, and biosafety level 4 is

required for work with dangerous agents that pose

a high risk of aerosol-transmitted laboratory infec-

tions. Currently all microorganisms expected to be

encountered in a food laboratory can be handled

with biosafety level 2 in the USA. Some countries

may require biosafety level 3 precautions when

working with E. coli O157:H7. In addition, biosafety

level 3 precautions are indicated for Clostridium

botulinum when activities pose a high potential

for aerosol or droplet production and for those activ-

ities involving large quantities of toxin.

Sample Handling – Hazards

0005Laboratory personnel can be exposed to infectious

agents through a number of routes (Table 1).

Inhalation Hazards

0006The generation of droplets and aerosols is considered

to be the primary means of laboratory exposure to

infectious agents. All laboratory procedures should

be performed carefully to prevent the creation of

aerosols and droplets. In most instances use of

proper containers and equipment will minimize the

chance for aerosolization of infectious materials.

However, even trivial laboratory operations generate

droplets and aerosols: a drop falling off the end of a

pipette, streaking the surface of a rough agar plate

with a loop, inserting a hot loop into a culture,

pulling a cotton plug from a flask or a tube, and

many others.

0007Pipettes should be drained gently with the tip

against the inner wall of the receiving tube, bottle,

or flask. The liquid should not be expelled forcibly

from the pipette. Material should be centrifuged in

sealed tubes or using centrifuges with sealed buckets,

safety trunnion cups, or sealed heads to prevent the

escape of aerosols and material being centrifuged.

Only sealed tubes should be used for vortex mixing.

Blenders should have leak-proof bearings and tight-

fitting gasketed lids. Sonication or homogenization of

infectious materials should be done inside a safety

cabinet.

0008Molds pose a potential inhalation hazard, as spores

may cause irritation. Moldy food should be sampled

with care, to minimize the potential for laboratory

contamination. When molds are grown on agar,

plates should not be inverted, as mold spores may

fall on to the lid of the Petri dish, and if it is opened

these can contaminate the laboratory.

tbl0001Table 1 Routes of exposure of laboratory personnel to

hazardous agents

Route Proceduresresultinginexposure

Contact Decanting liquids, pipetting, removal of screw

caps, vortex mixing, unsealed containers (spills)

Oral Mouth pipetting, not washing hands after working

with infectious materials

Ocular Procedures resulting in splashing, aerosol

generation (sonication, centrifugation,

homogenization)

Inoculation Use of needles, syringes

Inhalation Procedures generating aerosols (sonication,

centrifugation, blowing out pipettes, heating

inoculation loops; streaking inoculum on agar)

3444 LABORATORY MANAGEMENT/Microbiological Safety

Contact Exposure and Inoculation Hazards

0009 Infectious materials should be handled very carefully,

especially during sample transfers and decanting of

supernatants, to avoid spills. Safety glasses should be

used to protect against splashing infectious material

into the eyes.

0010 Because of the hazard of self-inoculation, the use of

needles and syringes should be restricted to pro-

cedures for which there are no alternatives. Needle-

locking syringes or disposable syringe-needle units in

which the needle is integral to the syringe should be

used. Needles should not be replaced in the guard

following use, nor should they be clipped.

0011 Care should be taken to avoid cuts when handling

sharp objects such as scalpels or broken glass. Broken

glass should not be picked up by hand; tongs,

forceps, or a brush and dustpan should be used.

Dispose of sharp objects in proper receptacles. In la-

boratories working with animals, training should be

given in animal-handling techniques to avoid bites and

scratches. Where appropriate, gloves should be worn.

Ingestion Hazards

0012 Mouth pipetting poses such a high risk of exposure

that good laboratory practices prohibit this pro-

cedure. Only mechanical pipetting devices should be

used in the laboratory.

0013 Additional ingestion hazards result from hand con-

tamination and subsequent transfer to the mouth; this

can be avoided by appropriate personnel practices

such as proper hand washing and wearing gloves

(see section on personnel practices and personal

hygiene).

Containment – The Biological Safety

Cabinet

0014 The most important element of containment is strict

adherence to good microbiological practices and

techniques. However, appropriate laboratory design

and safety equipment are also required for proper

containment of microbiological hazards.

0015 The use of proper containment equipment helps

minimize the exposure of laboratory personnel to

infectious agents. Equipment designed to avoid la-

boratory hazards includes biological safety cabinets,

mechanical pipetting devices, and centrifuges and

blenders. Containment is also achieved by use of

appropriate closures on laboratory glassware such

as test tubes, flasks, and bottles.

0016 The biological safety cabinet is the principal device

to provide containment of aerosols. There are three

types – class I, II, and III. Class I and II cabinets

are generally open-fronted and depend on air flow

to protect laboratory personnel.

0017The class I cabinet (Figure 1a) is designed to protect

the user; air enters the front work opening and exits

at the top through a high-efficiency particulate air

(HEPA) filter. This provides no protection against

contamination of the work materials, since unfiltered

ambient room air flows into the cabinet. A class I

cabinet should not be confused with a horizontal

laminar flow cabinet in which HEPA-filtered air is

blown across the work surface toward the researcher

to protect the research/materials against contamin-

ation. A horizontal laminar flow cabinet should

never be used with infectious agents due to potential

worker exposure.

0018Class II biological safety cabinets (Figure 1b)

protect the research materials from contamination,

as well as the worker, since both the supply and

the exhaust air are HEPA-filtered. Room air is

drawn through the front work opening into a plenum

below the work area by a fan. Air exiting the fan

is passed upwards through a channel in the rear into

a space between two HEPA filters. A portion of

the air is exhausted through one HEPA filter while

the rest of the air passes through the second HEPA

filter and is directed downward as clean, laminar flow

air to the work surfaces. In subclass IIA cabinets 30%

of the air is exhausted and 70% recirculated. In sub-

class IIB cabinets a smaller portion of air (30%) is

recirculated or there is total exhaust (no recircula-

tion). In the latter case room air entering through

the front opening to protect the researcher against

escaping pathogens is drawn into the plenum and is

exhausted.

0019The class III cabinet is a totally enclosed, airtight

workspace equipped with protective gloves, and

hence is often referred to as a glove box. Such cab-

inets are used for high-risk biological agents and are

not necessary in food microbiology laboratories,

although they are sometimes used for work with

Clostridium botulinum.

0020Biosafety cabinets should be located in a low-

traffic-area free from drafts. The cabinet must be

tested and certified at installation, after moving, and

annually thereafter. It is desirable that safety cabinets

exhaust outdoors since materials such as formalde-

hyde (used for decontamination), radioisotopes, or

chemical carcinogens should not be exhausted in-

doors. When cabinets are exhausted to the roof the

exhaust stack height should insure that discharge

occurs above the heads of maintenance personnel.

The exhaust stack should not exhaust in close prox-

imity to the air intake. HEPA filters should be dis-

posed of as contaminated biological waste, preferably

by incineration.

LABORATORY MANAGEMENT/Microbiological Safety 3445

Disinfection/Decontamination

Laboratory Waste

0021 Food laboratories generate potentially hazardous in-

fectious wastes during the analysis of samples con-

taminated by pathogens and during the propagation

of pathogenic microorganisms. Biological waste

containing viable microorganisms not known to be

hazardous to humans is not considered infectious

laboratory waste. Nevertheless, it is good laboratory

practice to decontaminate all waste containing viable

microorganisms and to disinfect work surfaces as if

working with infectious agents.

0022 Containment of waste Materials to be disinfected

or decontaminated prior to disposal or cleaning

should be placed in a centralized area designated for

biohazardous waste. A cart clearly labeled ‘To Be

Autoclaved’ is recommended for all laboratories in

which infectious organisms are handled. Tubes,

bottles, and flasks should be closed. Petri dishes

should be placed in trays or biohazard or autoclave

bags. Pipettes should be placed in pans or jars with

sufficient disinfectant to fill the pipette and cover it

completely. Materials to be disinfected and discarded

should be separated from those which are to be

cleaned and reused. Materials to be discarded can

be placed in autoclave bags which are sealed, auto-

claved, and then discarded.

0023Infectious waste (liquid and solid) should be decon-

taminated at the laboratory facility prior to transport

to a disposal site. If this is not possible, adequate

physical containment measures (sealed, leak-proof

containers) must be taken to prevent subsequent

exposure to those handling the waste.

0024Physical hazards Items with sharp points or edges,

such as needles, disposable pipettes, scalpel blades,

and broken glass, pose a special problem in decon-

tamination and disposal. These should be placed in

puncture-resistant containers either before or after

autoclaving. Needles and syringes should be placed

directly in the disposal container without replacing

the guard on the needle, since replacing the guard is a

frequent cause of inoculation of laboratory person-

nel. After decontamination, special containers that

provide a means to cut through both the needle and

the plastic syringe should be used to prevent reuse of

disposable needles and syringes.

HEPA

filter

HEPA

filter

HEPA

filter

Clean air

Contaminated

air

(a) (b)

fig0001 Figure 1 Biosafety cabinets: (a) class I and (b) class II. HEPA, high-efficiency particulate air. Reproduced from Furr AK (ed.) (1995)

CRC Handbook of Laboratory Safety, 4th edn. Boca Raton, FL: CRC Press, with permission.

3446 LABORATORY MANAGEMENT/Microbiological Safety

Disinfectants

0025 Disinfection/decontamination in a food laboratory

generally occurs by means of chemicals or by heat

(usually autoclaving). The type of treatment will

depend on the nature of the material to be treated,

the level and type of contamination, and the amount

of disinfection/decontamination required.

0026 Chemical Chemicals most frequently used for disin-

fection include quaternary ammonium compounds

(0.1–2%), chlorine compounds (0.01–5%), iodophor

compounds (0.5–1%), and alcohol (70–95%). Bio-

safety cabinets are sometimes decontaminated with

formaldehyde. Commercial disinfectants should be

used according to the manufacturer’s directions. Dis-

infection will depend on selection of the appropriate

chemical for the intended use, contact time, concen-

tration, and the presence of interfering substances.

For example, although 200 p.p.m. available chlorine

is generally adequate for disinfection, organic mater-

ials may be present which will combine with chlorine

and reduce its effectiveness. Thus it is recommended

that discard jars contain 1000 p.p.m. available chlor-

ine; 5% bleach (50 000 p.p.m. chlorine) is recom-

mended for cleaning spills.

0027 Heat Autoclaving is the method of choice for de-

contaminating cultures, glassware, and pipettes.

Autoclaving for 60–90 min at 121



C (15 psi),

depending on the loading conditions, to achieve a

waste temperature of at least 115



C for 20 min is

recommended. Effective autoclaving will depend on

time, temperature, and steam penetration. Tubes,

bottles, flasks, and bags should be loosely closed to

allow steam penetration. Tubes should be autoclaved

in racks. Glassware should be arranged to allow

steam to circulate around the individual containers.

The autoclave should never be overloaded. Contam-

inated glassware should contain liquids to facilitate

the sterilization process. Care should be taken when

adding water to avoid generating aerosols containing

infectious microorganisms. Stainless-steel pans, glass,

and heat-resistant plastic can be autoclaved. Metal

containers enhance transfer of heat to the waste

load, whereas plastic retards steam penetration.

Thus with certain materials or with larger volumes

of liquids it may be necessary to decrease the size of

the load or to increase time to compensate for poor

heat transfer/steam penetration. Autoclaved waste

can subsequently be disposed of as general waste.

0028 Dry heat may be used to decontaminate some

materials, including glassware and instruments, and

is recommended for anhydrous materials such as

oils, greases, and powders. These should be heated

at 160–180



C for 180–240 min.

0029Large volumes of infectious wastes, animal car-

casses, and contaminated bedding materials should

be incinerated. Since most incinerators are off-site,

solid waste must be contained in sturdy bags or

boxes and liquids in leak-proof containers for

transport. Trained personnel should transport the

containers to the incinerator.

0030Inoculating loops are sterilized by heat. Although

electric loop sterilizers are available, loops are gener-

ally sterilized by holding in a flame until the loop is

red-hot. Inoculating loops should be flamed by heating

the shaft until the sample has heat-dried before

flaming the loop itself. (Alternatively, flaming can be

avoided by use of sterile disposable plastic loops.)

Laboratory Decontamination

0031Frequency Chemical disinfection is used for bench-

tops, incubators, furniture, and floors which may

become contaminated. Work surfaces should be de-

contaminated at least daily and preferably before and

after each use. Biosafety cabinet surfaces should be

disinfected before and after each use. Other areas

should be cleaned and disinfected at least weekly.

Immediate cleaning and disinfection are required

when spills occur. Chemicals are also used in disin-

fecting items placed in discard jars; chemical solu-

tions should be replaced as often as necessary to

maintain an effective concentration of disinfectant.

0032Spills Spills of biohazardous materials require

special considerations. Spills can be contained by

using enamel or stainless-steel trays as a work surface.

If a spill occurs, the entire tray can be autoclaved.

Small spills can be cleaned up by pouring a concen-

trated disinfectant around the edges of the spill and

then covering the spill with disinfectant-soaked paper

towels. Additional paper towels are used to wipe

up the area. Contaminated paper towels are then

transferred to a biohazardous waste container for

autoclaving. Larger spills, however, can generate sig-

nificant aerosols which should be allowed to settle for

approximately 30 min prior to decontamination. The

laboratory should be evacuated and closed during this

period. A laboratory worker wearing a lab coat,

gloves, and respiratory protection should then clean

up the spill with disinfectant and paper towels,

sponges, or mops as appropriate. Clean-up materials

should preferably be disposable. When there is a large

spill in a biosafety cabinet, special disinfection

measures may be needed to decontaminate the fan,

filters, and air-flow plenums. When infectious agents

are spilled on clothing, contaminated garments

(lab coats, shoes, etc.) should be removed and

decontaminated.

LABORATORY MANAGEMENT/Microbiological Safety 3447

Centrifuge Accidents

0033 If breakage occurs during centrifugation the centri-

fuge should be turned off and allowed to stand closed

for 30 min. If breakage is discovered after the centri-

fuge is opened, the lid should be replaced and the

centrifuge left closed for 30 min. Broken tubes, glass

fragments, buckets, trunnions, and the rotor should

be placed in an appropriate noncorrosive disinfectant

and held for an appropriate time (generally 1–24 h) or

autoclaved.

Personnel Practices and Personal

Hygiene

0034 The most important aspects of microbiological safety

in the laboratory rely on good laboratory practices by

the laboratory worker. Persons working with poten-

tially infectious materials should be made aware of

the hazards and properly trained in the proper

methods for safely handling such materials. Insuring

that laboratory personnel are properly trained and

informed of potential hazards is the responsibility of

the laboratory director.

0035 Access to the laboratory should be limited, espe-

cially when working with infectious agents, and

doors should be kept closed when experiments are

in progress. As noted previously, mouth pipetting

should be prohibited. Eating, drinking, smoking,

handling contact lenses, and applying cosmetics

should not be permitted in the work area. Food

should be stored in cabinets or refrigerators desig-

nated for this purpose only and food storage areas

should be located outside the work area. Drinking-

water fountains should also be located outside the

work area. Workers should wear laboratory coats or

other protective covers. In some cases protective

gloves should also be worn. Open-toed shoes or

sandals should not be worn in the laboratory. Safety

glasses should be worn when filling syringes or when

handling certain chemicals such as strong acids. Lab

coats and gloves should be removed when leaving

the laboratory. Workers should wash their hands

following all laboratory activities, after removing

laboratory coats and gloves, and immediately

following contact with any infectious materials.

Workers should develop the habit of keeping their

hands away from their face, especially the mouth,

nose, and eyes, to prevent self-contamination and

infection. This is particularly important when

working with agents of low infectious dose, such as

E. coli O157:H7, where fewer than 10 organisms

have been shown to cause illness.

0036 Some employees may have personal health

conditions that place them at increased risk in the

laboratory, especially when working with certain

organisms. For example a pregnant woman may be

at increased risk from Listeria monocytogenes. Thus

most food laboratories do not allow pregnant women

to work in a laboratory where L. monocytogenes is

being handled. Persons who are immunocomprom-

ised may be at increased risk not only from L. mono-

cytogenes, but also from opportunistic pathogens

that may be encountered in foods. Careful assessment

of the risk of exposure should be made. If the expos-

ure cannot be eliminated or reduced, consideration

should be given to changing jobs. The employee must

be informed of the risk and involved in the decision-

making process.

0037Workers with open sores or cuts should not be

allowed to handle infectious materials that could

potentially contaminate the wound. If sores or cuts

are on the hands, bandages and gloves must be worn

to protect the worker.

0038For some infectious agents vaccines that provide an

extra level of protection may be available. However

for most foodborne disease organisms, vaccines are

either not available (e.g., Shigella spp., L. mono-

cytogenes, Campylobacter spp., most Salmonella

spp.) or only provide partial protection of short dur-

ation (e.g., Vibrio cholerae). A pentavalent (ABCDE)

botulinum toxoid is available through the US Centers

for Disease Control and Prevention as an investiga-

tional new drug and is recommended for personnel

working with cultures of C. botulinum or its toxins.

Individuals receiving the toxoid may experience

minor side-effects, including redness and swelling at

the site of the inoculation; in rare cases more general-

ized reactions have occurred. Hepatitis A vaccine is

available; although laboratory-acquired infection

with this virus is not considered a significant risk,

the vaccine is recommended for persons who work

in laboratories conducting research on this virus.

Other Considerations

0039All samples and cultures containing infectious mater-

ials should be labeled with the universal biohazard

symbol and the hazard identified on the label.

0040Foods for analysis that may be contaminated with

pathogens should be assumed to be contaminated and

handled appropriately. For example, swollen cans

may be contaminated with C. botulinum and should

be handled, using appropriate safety handling prac-

tices, by an individual who is immunized against the

toxins. Consideration should be given to the need to

open the cans in a safety cabinet.

0041Laboratories working with infectious agents

should have posted on the door the universal biohaz-

ard symbol identifying the infectious agent. The sign

should also list the name and phone number of the

3448 LABORATORY MANAGEMENT/Microbiological Safety

laboratory supervisor or other responsible party and

any special requirements for entering the laboratory.

Laboratories dealing with infectious materials should

be restricted to authorized persons only.

0042 Hazardous materials should be stored in locked

cabinets, refrigerators, or other storage areas and

access should be limited to authorized persons only.

0043 Food microbiology laboratories should have safety

guidelines posted or a safety manual located in an

accessible location. Safety practices appropriate for

the specific microbiological hazards that may be en-

countered should be included, as well as information

on chemical and physical hazards.

Summary of Microbiological Safety

Practices

1.0044 Avoid mouth pipetting.

0045 Handle samples in a manner that minimizes aero-

sol generation; for procedures known to create

aerosols, take steps to contain aerosols.

0046 Avoid the use of needles and syringes wherever

possible.

0047 Decontaminate work surfaces daily and after

spills.

0048 Keep laboratory doors closed when experiments

are in progress.

0049 Do not eat, drink, smoke, handle contact lenses, or

apply cosmetics in the laboratory.

0050Wear a lab coat when working in the laboratory

and remove it when leaving the lab.

0051Wash hands when leaving the laboratory and upon

contact with infectious materials.

See also: Cleaning Procedures in the Factory: Types of

Disinfectant; Hazard Analysis Critical Control Point;

Laboratory Management: Chemical Safety;

Microbiology: Classification of Microorganisms

Further Reading

Fleming DO, Richardson JH, Tulis JJ and Vesley D (eds)

(1995) Laboratory Safety: Principles and Practices, 2nd

edn. Washington, DC: ASM Press.

Furr AK (ed.) (1995) CRC Handbook of Laboratory Safety,

4th edn. Boca Raton, FL: CRC Press.

National Research Council (US) Committee on Hazardous

Biological Substances in the Laboratory (1989) Bio-

safety in the Laboratory. Washington, DC: National

Academy Press.

Richmond JY and McKinney RW (eds) (1999) Biosafety

in Microbiological and Biomedical Laboratories,

4th edn. US Public Health Service, Centers for Disease

Control and Prevention and National Institutes of

Health, HHS publication no. (CDC) 0093–8395. Wash-

ington, DC: US Government Printing Office.

LACTATION

Contents

Human Milk: Composition and Nutritional Value

Physiology

Human Milk: Composition and

Nutritional Value

C M Donangelo and N M F Trugo, Universidade

Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Introduction

0001 It is well recognized that human milk is the most

appropriate source of nutrition for infants during

the first 4–6 months of life. Human milk provides a

balanced composition of nutrients as well as many

types of bioactive components which have an import-

ant role in infant growth and development. Benefits of

breast-feeding for the infant include adequate

nutrition, protection against disease, development of

gastrointestinal and immune functions, and psycho-

logical well-being. It has been recognized recently that

breast-fed infants may have an advantage over for-

mula-fed infants as regards long-term cognitive devel-

opment and lower rates of neurological disorders.

0002Human milk is a very complex biological fluid that

contains numerous constituents distributed into sev-

eral physicochemical compartments: aqueous phase

(mineral ions, lactose, citrate, amino acids, whey

proteins, water-soluble vitamins), colloidal dispersion

LACTATION/Human Milk: Composition and Nutritional Value 3449

(casein micelles), emulsion (fat globules, membrane-

bound compounds, fat-soluble vitamins), and cells

(macrophages, neutrophils, lymphocytes, epithelial

cells). This compartmentation of human milk con-

stituents affects binding properties and bioavailabil-

ity to the infant. Interactions among components in

the compartments may influence the accessibility and

flow of nutrients to the infant as well as transmission

of biochemical messages to the infant via milk.

0003 Human milk composition shows marked inter- and

intraindividual variation. Several factors are known

to affect composition (Table 1). Stage of lactation is

responsible for the largest variation. Remarkable

changes in composition occur as lactation progresses

from the first few days (colostrum) to over 3 weeks

(mature milk) after childbirth. Typical values of

selected nutrients in colostrum and mature human

milk are shown in Table 2. Nutrients such as protein,

sodium, and zinc are present at higher concentrations

in colostrum while others, such as lactose and fat, are

present in higher concentrations in mature milk. After

the first month postpartum, milk composition gener-

ally stabilizes and further changes are less dramatic. It

has been proposed that alterations of nutrient supply

as lactation progresses reflect the changing nutrient

requirements of the infant, with a marked decrease in

growth rate over time, and also the development of

the infant’s capability to digest and absorb nutrients.

0004 Maternal diet is an important factor affecting the

nutrient composition of milk but the influence varies

in magnitude between nutrients. Concentrations of

individual fatty acids and of fat-soluble and water-

soluble vitamins generally reflect maternal intake.

Concentrations of lactose and minerals such as

sodium, calcium, phosphorous, magnesium, and iron

do not vary with maternal diet. Maternal hormonal

adjustments and nutritional stores may contribute

to prevent fluctuations depending on the nutrient so

that moderate dietary deficiencies or excesses are

not always reflected in milk composition. Severe

maternal malnutrition may, however, impair milk

volume and milk composition.

0005Several components of human milk, either nutri-

ents or nonnutrients, exert nonnutrient actions which

benefit the health and nutrition of the recipient infant.

tbl0002Table 2 Nutrient concentrations in human colostrum and

mature milk

Nutrient Colostrum

(1^5 days

postpartum)

Mature milk

(1^6 months

postpartum)

Macronutrients

Protein (gl

1

) 11–32

8–13

19–54

8–19

Lactose (gl

1

) 20–58

68–73

42–64

50–84

Fat (gl

1

) 10–28

36–43

27–40

Major minerals

Sodium (mmol l

1

) 17–22

4.7–8.0

7–12

5.2–10.4

Potassium (mmol l

1

) 17.5–18.5

10.4–13.9

7.2–15.1

9.5–15.9

Calcium (mmol l

1

) 5.4–8.0

4.7–7.5

5.1–11.6

3.5–9.0

Magnesium (mmol l

1

) 1.0–2.2

1.2–1.7

1.0–1.8

1.2–1.5

Phosphorus (mmol l

1

) 3.5–4.7

4.4–4.7

2.8–6.8

Trace minerals

Iron (mg l

1

) 0.6–1.0

0.3–0.9

0.4–1.2

0.3–2.2

Zinc (mg l

1

) 8–12

1–3

2.2–10.4

0.8–4.1

Copper (mg l

1

) 0.5–0.8

0.2–0.4

0.1–0.5

0.2–0.5

Manganese (mgl

1

) 5–12

3–6

Selenium (mgl

1

) 14–83

6–28

10–21

Fat-soluble vitamins

Vitamin A (retinol mg l

1

) 0.34–5.0

0.19–1.12

0.24–1.18

Vitamin K (mgl

1

) 1.8–5.2

1.2–9.2

Vitamin E (a-tocopherol mg l

1

) 8–22

1.8–3.4

Water-soluble vitamins

Ascorbic acid (mg l

1

) 44–60

19–41

20–105

Thiamin (mgl

1

) 56–200

100–160

160–238

Riboflavin (mgl

1

) 140–300

190–280

475–580

Vitamin B

(mg l

1

) 0.02

0.06–0.12

0.07–0.31

Folate (mgl

1

) 10–40

2–137

79–133

Vitamin B

(ng l

1

) 260–1200

78–170

160–970

Data from:

Kunz et al. (1999); and

Atkinson et al.,

Casey et al.,

Canfield

et al.,

Lammi-Keefe,

Picciano,

Prentice (data from outside Europe and

the USA) In: Jensen (1995) Handbook of Milk Composition, San Diego:

Academic Press.

tbl0001 Table 1 Factors affecting human milk composition

Stage of lactation

Changes during nursing

Diurnal rhythm

Gestational age at birth (preterm versus term)

Maternal diet

Maternal weight and body composition

Maternal disease (infections, metabolic disorders)

Maternal age

Parity

Geographic area

Ethnicity ?

Individuality

3450 LACTATION/Human Milk: Composition and Nutritional Value

They can be classified in several categories, according

to their actions. Since many of these bioactive sub-

stances present multiple roles and act synergistically

with each other, a particular component may be pre-

sent in more than one category. A broad category is

that of the defense agents, comprising cellular elem-

ents (leukocytes) and a variety of heterogeneous sol-

uble compounds, which may act directly or indirectly

against pathogens (antiinfectious agents) and/or alter

manifestations of infection due to their antiinflam-

matory properties (antiinflammatory factors), and

modulation of the immune responses (immunomodu-

lators). Other categories of beneficial compounds

present in human milk are growth factors and hor-

mones, which promote growth and maturation of

organ systems; binding proteins for hormones, vita-

mins, and minerals, which protect and increase the

bioavailability of the ligands; and several enzymes,

which ensure a better utilization of nutrients and act

as defense agents.

Nutritional Components of Human Milk

Protein

0006 Proteins are present in human milk as whey proteins

(50–90% of total), casein (10–50%) and milk fat

globule membrane proteins (1–3%). Whey proteins

include a-lactalbumin, the major protein in this frac-

tion (10–20% of total milk protein), serum albumin,

immunoglobulins, and lactoferrin. Casein is mainly

composed of two proteins: b-casein (85%), with dif-

ferent phosphorylated forms, and k-casein (15%), a

highly glycolysylated protein containing 40–60%

carbohydrate residues (hexoses, hexosamines, and

sialic acid). Casein micelles are formed by aggrega-

tion of caseins, calcium phosphate, and other ions,

depending on both hydrophobic interactions and

electrostatic binding. Human milk casein micelles

are considerably smaller (30–75 nm in diameter)

than bovine casein micelles (about 50–600 nm, mean

120 nm).

0007 The total protein concentration in milk is high

during early lactation (colostrum) and gradually de-

clines to a low level in mature milk (Table 2). Protein

concentration during early lactation is usually higher

in preterm than in term milk. There are pronounced

changes in the protein composition of milk as lacta-

tion progresses. Whey proteins decrease while casein

increases in milk during the first few days of lactation.

The ratio of whey proteins to casein is high (about

90:10) in colostrum and declines to 50:50 in mature

milk. The pattern in caseins is different for premature

milk, colostrum, and mature milk. In general, k-

casein is detectable in human milk only 3–4 days

postpartum and may not be detectable in preterm

colostrum. b-Casein concentration is higher in colos-

trum and mature milk of mothers with term com-

pared to preterm infants.

0008Milk proteins are a source of peptides, amino acids,

and nitrogen for the growing infant. In addition, milk

proteins serve several nonnutritive roles. Proteins in

human milk are not completely nutritionally available

to the infant. Proteins with nonnutrient functions must

remain intact in the infant gastrointestinal tract and

may be largely unavailable for nitrogen metabolism,

particularly during early lactation. However, at

6 weeks postpartum, 95% of breast milk protein is

considered nutritionally available in term infants.

Casein in human milk is an easily digested protein

which provides amino acids, calcium, and phosphorus

to the newborn. Casein phosphopeptides in human

milk have been shown to keep calcium (and probably

also bound iron, zinc, copper, and manganese) in sol-

uble form in the infant intestinal tract, thus favoring

absorption. a-Lactalbumin in human milk has a very

high nutritional value (protein quality) and its amino

acid composition is similar to the estimated amino acid

requirements of young infants.

Nonprotein nitrogen

0009The nonprotein nitrogen (NPN) fraction of human

milk comprises 20–25% of the total nitrogen and

remains relatively constant during lactation. This

fraction consists of more than 200 compounds,

including free amino acids, urea, uric acid, ammonia,

carnitine, choline, taurine, amino sugars, amino alco-

hols, nucleic acids, nucleotides, and polyamines.

Many of these components, such as taurine, choline

and nucleotides, are considered conditionally essen-

tial for the young infant. For example, human milk is

a source of purine and pyrimidine bases as well as

preformed nucleotides that are required by young

infants due to their high need of nucleic acid forma-

tion but limited capacity for de novo synthesis of

nucleotides.

0010Components of human milk NPN that contribute

to infant protein metabolism include some peptides,

free amino acids and, mainly, urea. Urea nitrogen is a

significant part (30–50%) of total NPN in human

milk, and it may contribute to infant protein synthe-

sis. Free amino acids represent 3–5% of the total

amino acids in human milk with higher levels in

colostrum than in mature milk. Glutamate/glutamine

and the nonprotein sulfonic acid, taurine, are the

predominant free amino acids in mature human

milk. Levels of urea and free amino acids in human

milk seem to reflect protein quality and protein intake

in the maternal diet. Increased urea and free amino

acid levels have been observed in women receiving

LACTATION/Human Milk: Composition and Nutritional Value 3451

high-versus low-protein diets. In mothers consuming

diets low in lysine, methionine, and/or tryptophane,

milk levels of these amino acids in the free form are

also low.

Carbohydrates

0011 Lactose is the major carbohydrate and, after water, is

the second major constituent in human milk (c.

70 g l

1

in mature milk). Other carbohydrates in

human milk include nucleotide sugars, glycolipids,

glycoproteins, and oligosaccharides, many of which

have several nonnutrient functions. Total oligosac-

charide content in human milk is 5–8gl

1

. Low levels

of free galactose and minor levels of glucose are also

found in human milk (c. 3 and 0.3 g l

1

in mature

milk, respectively).

0012 Lactose is one of the most stable constituents of

human milk, with variation in levels reflecting mainly

heterogeneity of individuals. Levels in milk increase

(slightly) during the first few days postpartum but

remain quite stable in mature milk (Table 2). Its

concentration decreases during nursing but no vari-

ation occurs if levels are corrected for the increase in

fat. Lactose levels may be low in preterm colostrum

and in colostrum of women with insulin-dependent

diabetes mellitus. Lactose in human milk is readily

available to the infant and has been reported to stimu-

late the absorption of calcium, magnesium, and

manganese.

Lipids

0013 Human milk lipids are the major energy source for

the infant, as well as a source of essential fatty

acids (linoleic, 18:2 n-6 and a-linolenic, 18:3 n-3),

and their long-chain polyunsaturated fatty acid

metabolites such as arachidonic acid (20:4 n-6), eico-

sapentaenoic acid (20:5 n-3), and docosahexaenoic

acid (22:6 n-3).

0014 Lipids are present in human milk in the form of

globules with a hydrophobic core consisting of tri-

acylglycerols, cholesteryl esters, and retinyl esters and

other neutral lipids, coated with bipolar compounds,

phospholipids, proteins, cholesterol, and enzymes,

formed into a loose layer called the milk lipid globule

membrane. The diameter of the globules ranges from

1to10mm, with most of the globules at 1 mm, giving a

large surface area for efficient binding to lipolytic

enzymes, thus facilitating lipid digestion and absorp-

tion in the infant gastrointestinal tract.

0015 Triacylglycerols account for at least 98% of

the milk lipids, followed by phospholipids (0.7%),

cholesterol (0.5%), and minor amounts of free

fatty acids, and mono- and diacylglycerols. The

major fatty acids in triacylglycerols are 18:1 and

16:0 (c. 30% and 20% of total, respectively). The

structure of the triacylglycerols in human milk is

unique with most of the 16:0 at sn-2, 12:0 at sn-3,

18:0 at sn-1, and 18:1 and 18:2 at sn-1 and sn-3.

Structure is an important factor controlling products

formed by the action of lipases and their intestinal

absorption in the infant.

0016Lipids are the most variable constituents of

human milk. Total milk fat increases with the stage

of lactation (Table 2) and during nursing; it decreases

with parity, maternal infection, and long-term mater-

nal undernutrition; it shows diurnal rhythm, and may

vary with maternal diet and body composition (adi-

posity). With the increase in total milk fat during the

first month of lactation, there is an increase in the

average size of the milk fat globules and a reduced

ratio of phospholipids and cholesterol (membrane

lipids) to triacylglycerols (core lipids).

0017A major proportion of human milk fat is formed

from circulating lipids derived from maternal diet,

and from maternal body stores. A small proportion

is synthesized de novo in the mammary gland from

glucose, primarily as saturated fatty acids with

medium-chain length (10–14 carbon atoms). Both

maternal diet and maternal adipose tissue affect the

composition of human milk triacylglycerols.

0018Maternal diet has a considerable influence on the

fatty acid composition of human milk. Long-chain

polyunsaturated fatty acids (such as 20:4 n-6, 20:5

n-3 and 22:6 n-3) which are synthesized by the

maternal organism from their essential precursors

(18:2 n-6 and 18:3 n-3), respond to changes in the

diet. For example, levels of eicosapentaenoic acid

(20:5 n-3) and docosahexaenoic acid (22:6 n-3) in

human milk are increased by consumption of fish

such as herring, mackerel, and salmon. The contents

of trans isomers of fatty acids in milk reflect maternal

intake of partially hydrogenated food fats and oils,

deep-fried foods, and dairy products.

0019Typical western and nonwestern diets affect the

fatty acid composition of human milk (Table 3).

On high-fat (western) diets, fatty acid composition

reflects dietary fatty acid pattern with high levels

of unsaturated fatty acids related to consumption of

vegetable oils. In this condition, only about 10% of

the milk fatty acids are synthesized by the mammary

gland. On high-carbohydrate low-fat (nonwestern)

diets, the proportion of fatty acids originating in the

mammary gland increases, resulting in higher levels

of medium-chain fatty acids in milk. On low-energy

diets, much of the milk fat is derived from adipose

tissue and the composition of milk triacylglycerols

reflects maternal depot fat. Maternal diet does not

affect total cholesterol and phospholipids levels of

milk.

3452 LACTATION/Human Milk: Composition and Nutritional Value