The document outlines essential equipment required for an embryology laboratory, including various microscopes, incubators, and specialized tools for sperm preparation and embryo analysis. It highlights the functionalities and applications of each piece of equipment, emphasizing their importance in maintaining optimal conditions for gamete and embryo handling. Additionally, the document addresses the use of advanced technologies such as time-lapse monitoring and laser-assisted techniques for enhanced embryo assessment.

1. IVF LAB INSTRUMENTS
REPALLE DEEPTHI
IVF LAB DIRECTOR
MOHAK IVF
SAIMS, INDORE

2.  Basic essential equipment required for
embryology laboratory includes the following.
 ADD-ONS
INTRODUCTION

3.  MICROSCOPES- Stereo zoom, phase contrast
and inverted microscopes.
 INCUBATORS-Incubator with accurately
regulated temperature and CO2/ trigas.
 Centrifuge for sperm preparation
 Heating stages / Heating blocks
 Refrigerator
EQUIPMENTS

4.  Micromanipulator
 Laminar flow cabinets
 Sperm counting chamber---Makler chamber or
hemocytometer
EQUIPMENTS

5.  ADD-ONS
 Programmed cell freezer & cryo preservation
unit.
 Thermocouple, CO2 gas analyzer, ph. meter,
VOC Meters- QA AND QC
 LASER- Assisted hatching, embryo biopsy
 IMSI- sperm morphology assessment
 TIMELAPSE- for continuous monitoring of the
embryso
EQUIPMENTS

6. Proper visualization of
gametes and embryos
are essential in an
embryology laboratory.
MICROSCOP
ES
1. Phase contrast microscope
2. Stereozoom Microscope
3. Inverted microscope

7.  Phase contrast microscope :
 Contrast enhancing optical technique.
 High contrast images of living cells in their
natural state.
 Without being previously killed, fixed or stained.
 APPLICATIONS:
 Semen analysis with a Makler chamber.
 The motility and morphology of the sample
along with concentration.
Phase contrast microscope

8.  PRINCIPLE:
 It uses a conventional light microscope fitted
with a phase contrast objective and phase
contrast condenser.
 The technique is based on the fact that light
passing through one material to another material
of a slightly different refracting index (thickness)
will undergo a change in phase.
 These differences in phase are translated in to
variation in brightness of a structure.
Phase contrast microscope

9.  Stereozoom Microscope :
 This provides a spatial, three dimensional image
of the object.
 The working distance is inversely proportional to
the magnification.
 Usually an objective magnification of 1 – 1.5 x is
used.
 APPLICATIONS:
For scanning the follicular aspirates and for
identification of oocytes and embryos.
Stereozoom Microscope

10.  PRINCIPLE:
 The stereomicroscope takes advantage of its
ability to perceive depth by transmitting twin
images that are inclined by a small angle
(between 10 & 12°) to yield a true stereoscopic
effect.
 Zoom system provide a continuously variable
magnification range while simultaneously
keeping the microscope in focus.
Stereozoom Microscope

11.  Inverted microscope :
 Micromanipulation is conducted using an
inverted microscope between 200 and 400 x, and
therefore 20 and 40x objectives are essential.
 Procedures Interference optics like Hoffman
modulation contrast, Normarski are preferred as
they permit the best measure of detail and depth.
 APPLICATIONS:
 For detailed examination of oocytes and embryos
and micromanipulation
Inverted microscope

12.  A stage heater on the microscope is essential to
maintain temperature in culture dishes for all
procedures.
 Each microscope should be equipped with a still
camera or video camera with monitor which will
help in image storing and education purpose.
Inverted microscope

13.  Two incubators with accurate CO2 and temperature
controls and a table top incubator.
 One for sperm work and equilibration of media.
 Second for culture of oocytes and embryos.
 Incubation with separate compartments – like six door
incubator allow minimal disruption of P.H of culture
media and temperature.
 Either 6% CO2 in gas phase or triple gas mixture of 6%
CO2 / 5% O2 / balance N2 can be used.
INCUBATOR

14.  Outer casing is made of electrolytically
galvanized steel sheet.
 Control elements are made of temperature
resistant plastic. Inner casing is of stainless
steel.
 A platinum point 100 resistance serves as
temperature sensor.
 Co2 sensor- TC or IR.
INCUBATOR

15.  Principle:
 An air jacket heating system heats the interior.
 Outer door is also heated so that there will not be any
condensation on the glass door.
 The sterile water kept in the reservoir evaporates and
humidifies the chamber atmosphere.
INCUBATOR

16.  The incubator temperature should be at least
8°c above the ambient temperature.
 The relative humidity inside the chamber should
be >95%.
 The water used should not contain agents which
will corrode the reservoir.
INCUBATOR

17.  GAS SUPPLY :
 Gas will pass through sterile filters where particles larger
than 0.3µm are retained, filter efficiency is 99.99%.
 Small fan – proper spreading of gas throughout the
incubator.
 Opened door, the switch cuts off the gas supply and
heating system – alarm indication.
 The outer door is not able to close, unless the inner glass
doors are shut air tight – alarm indication.
INCUBATOR

18.  For sperm preparation and concentration of
sperms.
 There are two types of centrifuges
1. Manual
2. Digital
CENTRIFUGE

20.  Heating stages - To keep specimens at 37°c.
 Heated stage on micromanipulation microscope
and on stereo microscope.
 PITFALLS:
 Hot spots on stages can exceed critical
thresholds. Thermal cycling can be a problem,
to maintain 37°c, the stage may actually cycle
between 36 – 38°c.
HEATING STAGES

21.  To keep follicular aspirates warm at 37° until
oocyte identification, to keep culture tubes
warm for short period of time and warming
culture media with HEPES buffer.
 While doing the semen preparation for IUI and
ICSI.
HEATING BLOCK

22.  For bilateral manipulation with micro tool
holders, suction / injection and biopsy device.
 A micromanipulator is preferably placed on an
anti vibration table.
 Two basic types of manipulation systems –
 Motorized hydraulic - Narishige
 Mechanical - RI
 Robotic- Eppendorf
MICRO MANIPULATOR

23.  Pre filters and HEPA filters, remove the particulate
matter less then 0.3µm.
 Vertical flow one is usually used. It supply sterile air.
 Two cabinets are required –
 One for sperm preparation
 Second for setting up of culture dishes, examining
and grading oocytes and embryos and loading
embryos into catheters for transfer.
LAMINAR FLOW CABINETS

24.  For storage of media, chemicals and heparin.
 The temperature of the refrigerator should be
maintained at 4°c.
 All the medium should be maintained in
refrigerators under sterile conditions.
REFRIGERATOR

25.  Makler counting chamber is only 10µm deep,
1/10th
of the depth of ordinary hemocytometers.
 The cover glass has a 1mm2
fine grid in the center
subdivided into 100 squares of 0.1 x 0.1 m each.
 Spacing is secured by 4 quartz pins.
 5µl of well mixed undiluted specimen is placed in
the center of the chamber. A microscope objective
of 20x is required.
Makler chamber / Hemocytometer

26. 1. Dilution not necessary - analyzed quickly.
2. Applied spermatozoa are uniformly distributed
and monolayered.
3. Easy and more convenient than hemocytometer.
4. Errors incurred by uncontrolled pressure
applied to the coverslip are avoided.
5. Chamber is quickly and easily available for
reuse.
ADVANTAGES

28.  One Chamber consists of 25 large squares.
Each large square in surrounded by triplet lines
and contains 16 small squares. Each counting
chamber measures
 1mm x 1mm has a depth of 0.1mm [100µm]. So
the total volume in one chamber is 1 x 1 x 0.1 =
0.1 mm3
= 0.01µl = 10nl.
NEUBAUER IMPROVED HEMOCYTOMETER

29.  With supplies of liquid nitrogen, the embryos and
sperms can be frozen in a controlled program in the
cell freezers (kryo planner).
 Storage Dewar's with canisters suitable for efficient
and effective access to straws and ampoules are
used for long term storage of gametes.
PROGRAMMED CELL FREEZER

31.  For assisted hatching and PGD
LASER

32.  Time-lapse embryo incubation is an application
of time-lapse systems used in IVF where
embryos cultured in an incubator are
monitored with an advanced camera system.
It enables continuous observation without
disturbing the embryos' culture and
environment.
Timelapse

33.  IMSI-Intracytoplasmic
morphologically selected sperm
injection (IMSI) is a sperm
selection method used in
intracytoplasmic sperm
injection (ICSI). The technique
involves using a microscope to
view detailed images of the
sperm under very high
magnification (over x6000) to
select the sperm to inject into
an egg.
IMSI

34. 1. PH meter
2. Osmometer
3. CO2 gas analyzer
4. VOC meter
5. Air particulate counter
 Water purification system
 Sterlization system – Plasma sterilization,
autoclaving.
Other equipments

35. THANKS