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Curr Protoc Microbiol. Author manuscript; available in PMC 2021 Jun 1.
Published in final edited form as:
Marc Y. Fink,1,2 Danielle Shapiro,1 and Steven M. Singer1,3
Author information Copyright and License information PMC Disclaimer
The publisher's final edited version of this article is available at Curr Protoc Microbiol
Abstract
Giardia lamblia is a protozoan parasite that is found ubiquitously throughout the world and is a major contributor to diarrheal disease. Giardia exhibits a biphasic lifestyle existing as either a dormant cyst or a vegetative trophozoite. Infections are typically initiated through the consumption of cyst-contaminated water or food. Giardia was first axenized in the 1970s and can be readily maintained in a laboratory setting. Additionally, Giardia is one of the few protozoans that can be induced to complete its complete lifecycle using laboratory methods. In this article, we outline protocols to maintain Giardia and induce passage through its lifecycle. We also provide protocols for infecting and quantifying parasites in an animal infection model.
BASIC PROTOCOL 1: IN VITRO MAINTENANCE AND GROWTH OF GIARDIA TROPHOZOITES
BASIC PROTOCOL 2: IN VITRO ENCYSTATION OF GIARDIA CYSTS
BASIC PROTOCOL 3: IN VIVO INFECTIONS USING GIARDIA TROPHOZOITES
Keywords: Giardia lamblia, Giardiasis, murine infection, passage and maintenance, cryopreservation, animal model, protozoan parasite
INTRODUCTION
Human infection with Giardia lamblia (giardiasis) is regarded as one of the most common diarrheal diseases in the world with an estimated annual incidence of 280 million cases. Symptoms are characteristic of gastrointestinal distress; however, most cases are asymptomatic. Several studies indicate that subclinical Giardia infection is associated with childhood malnutrition. Paradoxically, another study suggests that the presence of this parasite may be protective against severe pediatric diarrheal disease. Adding to this complexity, long-term post-infectious syndromes in humans have been reported despite the eradication of this parasite.
Giardia lamblia (syn. G. duodenalis, G. intestinalis) is a flagellated protozoan microorganism that is found ubiquitously throughout the world. The first reported discovery of Giardia is attributed to Anton van Leeuwenhoek, the pioneer of microscopy when he examined his stool for microorganisms and was able to accurately describe this parasite’s features (Dobell, 1920). This article will focus on the growth and maintenance of Giardia lamblia (hereafter called Giardia) as this organism can be readily cultivated in a laboratory setting. This protocol has been optimized for the growth of G. lamblia, the only Giardia species that infects humans. G. muris is the natural rodent pathogen, but does not infect humans and is not readily cultivatable in axenic culture. Additionally, this article will not cover the isolation of Giardia from human clinical samples and will solely focus on culturing Giardia and infecting mice within a laboratory setting.
Giardia is considered to be a parasite since its replication and growth require the colonization of the human or mammalian intestinal tract (Adam, 2001). However, Giardia is not an intracellular pathogen and does not require the use of other cell types for expansion in vitro or in vivo. Instead, these cells adhere to surfaces and actively replicate via asexual binary fission if conditions are optimal. Thus, Giardia is readily capable of being cultivated and maintained indefinitely in a controlled laboratory setting as a monoculture using the methods described below.
The Giardia lifecycle has two distinct phases: a vegetative trophozoite and an infective cyst that is resistant to harsh environmental conditions. Cysts are found in most natural sources of water, such as rivers or lakes, however, cysts are more likely to be found in areas that are contaminated with animal fecal matter. Once cysts are ingested by a host, the excystation process begins after reaching the stomach. The acidity of the stomach triggers the breakdown of the Giardia cyst wall. In the small intestine, each cyst releases two excyzoites, which then generate four trophozoites following cell division. These trophozoites proceed to colonize the host’s intestinal tract, particularly the duodenum of the upper small intestine where bile is available to support growth and replication. Eventually, trophozoites initiate the encystation process and migrate towards the lower intestine where they are shed from infected hosts to the outside environment as infective cysts (Adam, 2001).
Giardia is divided into 8 genetic groups, termed assemblages A-H. Each assemblage is generally associated with particular host-specificity and most assemblages do not infect humans. The most relevant assemblages to human health are assemblages A and B since these two are the primary assemblages found in human giardiasis (). However, a small number of reports have described human infections with Giardia assemblages other than A and B (; ). Within these two human-infective assemblages: the WB strain (Assemblage A, ATCC #50803), the GS strain (Assemblage B, ATCC #50581), and H3 strain (Assemblage B, Waterbourne, Inc.) are three of the most commons strains used for in vitro and in vivo laboratory experiments. Giardia cultures are typically grown using the trophozoite form of this parasite as most strains of Giardia will actively divide in modified TYI-S-33 media (Keister, 1983); however, the growth rate of trophozoites varies between the strain of Giardia being cultivated. Doubling times range from 8–12 hours depending mainly on the strain. Trophozoites are primarily grown in borosilicate glass tubes, but cell culture flasks (T-25, T-75) can also be used if a large number of trophozoites are needed (e.g., infection of mice). During growth, the bulk of trophozoites will adhere to the interior surface of the culture vessel and will also swim in the supernatant of the media. Mechanical or enzymatic means are not required to detach adherent trophozoites as a simple chilling of the culture vessel will trigger the detachment of cells. Giardia cyst formation can also be induced from trophozoite cultures using media that mimic the biochemistry of the small intestine and contains higher concentrations of bile or higher pH levels. Both forms of Giardia must be cultivated at 37°C, but do not require additional humidity or 5% CO2 for growth. Strict anaerobic growth conditions are not necessary for cultivation since Giardia is considered a microaerophile and can tolerate small amounts of O2; however, if large amounts of O2 remain within the culture vessel, they will generate toxic compounds leading to a collapse of the Giardia culture. Trophozoites and media-generated cysts can be used in vitro to examine general Giardia biology or can be used in cell co-cultures with other mammalian cells to examine host cell responses.
CAUTION
Giardia sp. is a Biosafety Level 2 (BSL-2) pathogen. Giardia lamblia is capable of human infection and laboratory infections have been reported. Follow all appropriate guidelines and regulations for the use and handling of Giardia. Guidelines for biosafety and practice can be found in the latest edition of Biosafety in Microbiological and Biomedical Laboratories (Current edition: 5th Edition) and refer to section VIII-C: Parasitic Agents. See UNIT 1A.1 and other pertinent resources (APPENDIX 1B) for more information.
NOTE
All solutions and equipment coming into contact with living cells must be sterile and proper aseptic technique must be maintained.
NOTE
All culture incubations should be performed in a 37°C incubator unless otherwise specified. Humidity or 5% CO2 is not necessary for the growth of this parasite.
STRATEGIC PLANNING
BASIC PROTOCOL 1: IN VITRO MAINTENANCE AND GROWTH OF GIARDIA TROPHOZOITES
Giardia is an intestinal parasite that possesses a biphasic lifestyle comprised of a dormant cyst or vegetative trophozoite. The latter form is responsible for the colonization of the intestinal tract and directly contacts host cells. Accordingly, most cell culture models use Giardia trophozoites of strain WB, whereas murine infection models use either trophozoites of strain GS or cysts of strain H3. As noted above, WB is a member of assemblage A, and both GS and H3 are members of assemblage B. The preference for using WB trophozoites for in vitro work is mainly historical, although it is easier to perform transfection experiments using WB as plasmids replicate episomally in this strain, but not in GS (Singer, Yee, and Nash, 1998). However, WB trophozoites do not readily establish infections in adult mice. The preference for using GS trophozoites for infection in mice is mainly historical, while the use of H3 cysts is facilitated by the ability to purchase these commercially. Giardia is not an intracellular pathogen and does not require the use of other cell types for expansion in vitro or in vivo. Trophozoites are simple to cultivate within a laboratory setting and will expand in a short time. As Giardia is microaerophilic, trophozoites will not tolerate large amounts of oxygen and air-tight culture vessels that are devoid of additional air space must be used for culturing.
Materials (Unless specified, catalog numbers refer to Fisher Scientific, although other vendors are equally valid)
Giardia Media (Complete modified TYI-S33 medium) (see recipe)
Giardia freezing medium (see recipe)
70% Ethanol
Phosphate buffered saline (PBS) without calcium or magnesium
Frozen stock of G. lamblia GS clone H7 (ATCC 50581) or Luciferase-expressing trophozoites (e.g., Barash et al., 2017)
37°C Cell Culture Incubator
37°C Water or Bead Bath
Beckman Coulter Allegra X-30 refrigerated benchtop centrifuge (or equivalent) with appropriate 15- and 50-mL centrifuge tube adaptors
Inverted microscope equipped with phase-contrast optics
Hemocytometer
15 mL conical tube (Cat. #: 05–539-12) or equivalent
Fisherbrand disposable borosilicate glass tubes with threaded end 100mm (16 mL volume) (Cat. #: 14–959-35A)
Fisherbrand screw caps for disposable glass tubes (Cat. #: 14–957-82A)
Test-tube Slant Rack (e.g. Cat. #: 14–810N)
25-cm2 (T-25) cell-culture flasks with plug-seal caps
A bucket filled with ice
Sterile cryovials
Freezing container capable of 1°C/min cooling rate (e.g. Cat. #: 07–210-00).
−150°C freezer or liquid nitrogen tank
Thawing Trophozoites
Work in a biological safety cabinet (BSC) while wearing a protective laboratory coat and disposable examination gloves.
Warm complete modified TYI-S33 medium (Giardia media) to 37°C in water or bead bath.
Clean BSC working area with soap and water and then sterilize with 70% ethanol. After warming, sterilize the Giardia media container by wiping with 70% ethanol before placing it inside the BSC.
Transfer 10 ml of warm media to a sterile 15 ml centrifuge tube.
Remove a frozen cryovial of G. lamblia from −80°C freezer or liquid nitrogen tank and quickly place it into a 37°C water bath.
After the freezing vial has mostly (but not completely) thawed, spray the cryovial with 70% ethanol to avoid transferring microbial contaminants from the water bath and place it into a sterile and clean BSC.
Using a sterile pipette, transfer thawed Giardia cells from the cryovial into the 15 mL conical tube containing Giardia media. Close the tube and mix by inversion several times.
Centrifuge the tube at 600 × g for 10 minutes at 4°C and aspirate supernatant to remove the freezing medium containing DMSO.
Gently resuspend the pellet in 15 mL of warm Giardia media and transfer contents into a sterile borosilicate glass culture tube until liquid fills the entire vessel, leaving ~5 mm of air space. Close securely to prevent gas exchange. Be sure not to overfill the container with media. Any spillover must be cleaned with 70% ethanol to avoid microbial growth on the outside of the tube.
Alternatively, the Giardia suspension can be transferred into a T-25 flask rather than a culture tube. The flask will require ~55 ml of media.
When adding suspension into either a culture tube or flask, the media must fill the entire culture container to reduce the amount of oxygen within the vessel. As Giardia is a microaerophilic organism, large amounts of space (oxygen) within the container will lead to media oxidation and parasite death.
Label the tube with strain, initials, and the date and then place the tube in a 37°C incubator to grow overnight. Place tubes on a slanted rack 10–15˚ from horizontal so that any air bubbles maintain minimal surface area contact with the media.
The next day, warm Giardia media to 37°C in water or bead bath, sterilize BSC and Giardia media container and place media into BSC.
Check the tube for cell viability and growth using an inverted phase-contrast microscope.
Live Giardia trophozoites will adhere to the bottom of the tube and also may be swimming or “spinning” in the supernatant (despite flowing with the movement of the liquid). Under higher magnification, adherent cells can also be seen beating their flagella. Dead cells will be floating with the movement of liquid and will not show any movement.
Sterilize the Giardia culture tube with 70% ethanol and place it into BSC.
Aspirate media from tube to remove any residual DMSO and replace it with sterile, warm Giardia media. Be sure to use a secondary trap containing bleach to kill any trophozoites in the aspirate.
Incubate tube in 37°C incubator. Check the tube for cell viability, growth, and contamination using an inverted phase-contrast microscope daily until ready for passage or use. This is generally when trophozoites reach 70–80% confluence.
Trophozoites of different strains exhibit different growth rates. The assemblage A stain WB may double in ~ 8 hours, while the assemblage B strain GS doubles roughly every 12 hours.
Passaging and Expanding Trophozoites
Work in biological safety cabinet (BSC) while wearing protective laboratory coat and disposable examination gloves.
Warm complete modified TYI-S33 medium (Giardia media) to 37°C in water or bead bath.
Clean BSC working area with soap and then sterilize with 70% ethanol. After warming, sterilize the Giardia media container with 70% ethanol and then place it inside.
Remove a G. lamblia culture tube from 37°C incubator and examine for cell viability, growth, and contamination.
Place the tube in the ice bucket and cover completely with ice.
Let the tube chill for 15–30 minutes as trophozoites will start to detach as media cools.
Sterilize the outside of the culture tube with 70% ethanol and place it into BSC.
Transfer a small volume of detached cells to a new sterile glass tube and completely fill the tube with fresh Giardia media.
A 1:100 dilution will generally result in a new culture being ready for passage or use after 48–72 hours. This will vary based on the strain being used.
Label tube with strain, initials, and date and place tube in 37°C incubator to grow.
If expanding trophozoites to culture a greater number of trophozoites then transfer cells into a T-25 cell culture flask rather than a glass tube in step 23.
Harvesting Trophozoites
Work in BSC while wearing a protective laboratory coat and disposable examination gloves.
Clean BSC working area with soap and then sterilize with 70% ethanol.
Remove a G. lamblia culture tube or flask from the 37°C incubator and examine for cell viability, growth, and contamination.
Confluent cultures in flasks or tubes should have ~106 trophozoites/ml. Lower yields could mean that the reagents used for media production are not optimal. Testing specific lots of casein and serum, in particular, may be necessary. Low yields might also reflect insufficient time on ice to fully detach trophozoites, use of old oxidized media (generally obvious from the appearance of hexagonal crystals of cystine in the cultures), or trophozoite death due to incubating cultures too long before harvest.
Place the tube or flask in an ice bucket, cover completely, and let cells detach for 30–45 minutes. Flasks require more time than tubes to chill.
After cells have detached, sterilize the outside of the tube or flask with 70% ethanol and, in a BSC, transfer all media to 15 mL conical tubes. Be sure to properly equalize and balance tubes.
If using a T-25 cell culture flask, then upscale to 50 mL conical tubes (any sterile polypropylene tube should be fine) and adjust accordingly.
Centrifuge tubes at 600 × g for 10 minutes at 4°C to pellet trophozoites.
Aspirate supernatants, combine pellets (if necessary), and wash trophozoites with 14 mL of sterile PBS. If using cells for in vitro encystation then do not resuspend pellet into PBS and instead proceed to Basic Protocol 2.
For cells resuspended in a 50 mL conical tube, add >25 mL of PBS. After centrifugation, the pellet will still be somewhat “loose” within a 50 mL conical tube, so be sure not to suck up the pellet during aspiration.
Centrifuge trophozoites again at the same speed, temperature, and time. Wash pellets for at least 4 times.
If using trophozoites for in vitro co-culture with epithelial cells or immune cells, then be sure to wash trophozoites as described. Residual yeast extract from Giardia media is highly stimulatory and can induce cytokine release from cells on its own. Saving aliquots of the supernatant from the final wash provides good control for residual media contamination.
Count cells using a hemocytometer before the final centrifugation step. Resuspend cells in sterile PBS at the desired concentration. Infection of mice proceeds on Basic Protocol 3.
Alternatively, cells can also be resuspended in mammalian cell culture medium in preparation for co-culture experiments; however, media requirements for co-cultures will vary.
Freezing Trophozoites
Work in BSC while wearing a protective laboratory coat and disposable examination gloves.
Clean BSC working area with soap and then sterilize with 70% ethanol.
Prepare Giardia freezing media A and B (see recipes). Keep the freezing media chilled by placing media in an ice bucket.
A single T-25 flask will be able to generate enough cells to freeze 5 cryovials that each has a total volume of 1 mL.
The final freezing medium is essentially TYI-S33 with an extra 10% FBS and 10% DMSO. Freezing media should remain ice-chilled since combination with DMSO will generate an exothermic reaction that results in the increased media temperature and may potentially harm the cells during the freezing process. Pure DMSO should not be ice-chilled as it will solidify at 0˚C.
Remove a G. lamblia culture flask from the 37°C incubator and examine for cell viability, growth, and contamination. Trophozoites should be frozen when confluency reaches 70–80%.
Place the entire flask into an ice bucket to chill and let the flask chill for 45–60 minutes.
Label cryovials with strain, initials, and date of freezing and chill on ice.
After the flask has sufficiently chilled, sterilize the outside with 70% ethanol and place it into BSC.
Transfer all the media within the tube into 1–2 50 ml conical tubes.
Centrifuge tubes at 600 × g for 10 minutes at 4°C to pellet trophozoites. Be sure to properly balance tubes in a centrifuge.
Remove supernatant from pelleted cells and resuspend the pelleted cells in 2.5 ml Freezing medium A (without DMSO).
Add 2.5 ml Freezing Medium B and mix gently.
DMSO destabilizes the membranes of the trophozoites and vigorous pipetting can kill cells.
Aliquot ~1 mL of cells into each pre-cooled cryovial and transfer vials into a freezing container capable of cooling a 1°C/min within a −80°C freezer.
After cells have frozen, take a single cryovial and thaw trophozoites (step 1) to check for viability and contamination. If cells are viable and uncontaminated then move cryovials to a −150°C freezer or liquid nitrogen tank for long term storage and cryopreservation.
BASIC PROTOCOL 2: IN VITRO ENCYSTATION OF GIARDIA CYSTS
The two protocols listed in this article aim to induce encystation through a combination of increased pH adjustment increased bile concentration and the addition of lactic acid salts. Gillin, Boucher, Rossi, and Reiner (1989) were the first to establish a formalized protocol to generate a robust number of viable cysts in vitro by expanding on the observation that primary bile salts and a slightly basic pH were able to induce encystation (; Gillin et al., 1987). In this protocol, trophozoites are first starved of bile in a pre-encystation medium and then introduced to encystation medium #1 that contains porcine bile, lactic acid, and a more basic pH. The second protocol (encystation protocol #2) developed by Sun, McCaffery, Reiner, and Gillin (2003) incorporated excess bovine bile and is devoid of a formal pre-encystation medium other than Giardia media. This protocol requires less time to generate viable cysts and is more efficient for strain GS (). The initiation of cyst formation can be identified by the production of encystation specific vesicles (ESVs) 6–12 hours after the addition of either encystation media (Fig. 2). Alternately, encystation progress can be monitored using flow cytometry for DNA content. Trophozoites in G1 have 4N DNA content, in G2 they have 8N, and cysts should have 16N (). Additionally, viable cysts should form into typical cyst morphology, be resistant to water treatment, and also return into trophozoites after excystation. The quality of cysts derived from either protocol should be tested to determine the most appropriate protocol for a particular investigation. Neither protocol, as far as we are aware, can produce cysts capable of infecting animals. Cysts isolated from feces or purchased from a commercial supplier (i.e. H3 cysts from Waterbourne, Inc.) should be used for animal infections initiated by cysts.
Brightfield image of Giardia strain GS cultured in encystation medium #2 for 72 hours. A trophozoite is indicated by the red arrow, while green arrows indicate encysting cells with encystation vesicles.
Materials
Pre-Encystation Medium (see recipe)
Encystation Medium #1 (see recipe)
Encystation Medium #2 (see recipe)
Excystation Stage 1 Solution (see recipe)
Excystation Stage 2 Solution (see recipe)
Giardia Media (Complete modified TYI-S33 medium) (see recipe)
Deionized, distilled water (ddH2O)
37°C Cell Culture Incubator
37°C Water or Bead Bath
70% Ethanol
A bucket filled with ice
Logarithmic Giardia trophozoite culture in 12 mL glass culture tube
Beckman Coulter Allegra X-30 refrigerated benchtop centrifuge (or equivalent) with appropriate 15- and 50-mL centrifuge tube adaptors
Inverted microscope equipped with phase-contrast optics
Hemocytometer
15 mL conical tube
1.5 mL microcentrifuge tubes (e.g., Cat. #: 05–408-129)
Fisherbrand disposable borosilicate glass tubes with threaded end 100mm (12mL volume) (Cat. #: 14–959-35AA)
Fisherbrand screw caps for disposable glass tubes (Cat. #: 14–957-82A)
Encystation Protocol #1 (Gillin et al., 1989)
Prepare pre-encystation media and warm to 37°C in bead or water bath.
Work in BSC while wearing a protective laboratory coat and disposable examination gloves.
Clean BSC working area with soap and then sterilize with 70% ethanol.
Remove a G. lamblia culture tube from the 37°C incubator and examine for cell viability, growth, and contamination.
Prepare cells as described in Basic Protocol 1, Harvesting Trophozoites.
Pellet trophozoites and resuspend cells at a density of 5000 trophozoites/mL in pre-encystation media into a new clean, sterile glass culture tube or T-25 flask.
Culture cells for 3 days at 37°C.
After 3 days, prepare encystation medium #1 and warm it to 37°C.
Aspirate media and unadhered cells from the Giardia cultures in pre-encystation media.
Fill tubes or flasks with warmed encystation medium #1 until full to initiate the encystation process.
Culture for an additional 42–48 hours at 37°C.
Cyst production and recovery may vary with media and strain.
Check cultures for cyst formation and contamination.
Chill encysting cultures on ice for 10–20 minutes if using tubes and 30–45’ if using flasks.
Transfer media and detached cells to 15 mL or 50 mL conical tubes and centrifuge at 600 × g for 10 minutes at 4°C.
Resuspend pellets in 12 mL of sterile ddH2O to lyse trophozoites and place cells on ice for 10–20 minutes.
Cysts are water-resistant and will not lyse.
Spin as before and repeat water-treatment at least two more times.
After the last wash, count cysts with a hemocytometer and store at 4°C until use. In vitro cysts are not as stable as natural cysts and will lose viability quickly.
Encystation Protocol #2 (Sun et al., 2003)
Prepare encystation medium #2 and warm to 37°C in bead or water bath.
Work in BSC while wearing a protective laboratory coat and disposable examination gloves.
Clean BSC working area with soap and then sterilize with 70% ethanol.
Remove a G. lamblia culture from the 37°C incubator and examine for cell viability, growth, and contamination.
Aspirate Giardia media within a culture tube or flask and refill with warm encystation medium #2 to initiate the encystation process.
Culture tubes for 40–48 hours in 37°C incubator.
Cyst production and recovery may vary with media and strain. Using strain GS we obtain ~10% encystation rates with this protocol. (See Fig. 2).
Check cultures for cyst formation and contamination.
harvest and water-treat encysting cell cultures as described before (Encystation Protocol 1, steps 13–16).
Count cysts with a hemocytometer and store at 4°C until use.
Excystation Protocol ()
Warm excystation stage 1 solution to 37°C in bead or water bath.
Clean BSC working area with soap and then sterilize with 70% ethanol.
Place 2×106 type 1 cysts in a 1.5 mL microcentrifuge tube.
Type 1 cysts appear smooth and have an oval shape with clearly defined intracellular organelles.
Spin for 5 minutes at 4°C at 135 × g.
Discard the supernatant and resuspend pellet with 1.5 mL of stage 1 solution and briefly vortex to mix.
Incubate cysts at 37°C for 30 minutes.
Prepare excystation stage 2 solution and warm in 37°C bead or water bath along with Giardia media.
Alternatively, Tyrode’s salt solution (without trypsin) can be pre-warmed and can be mixed with trypsin right before use.
Briefly vortex incubating cysts.
Sediment cysts for 5 minutes at room temperature at 135 × g
Discard the supernatant and resuspend cells in 1.5 mL of stage 2 solution. Briefly vortex to mix cysts.
Incubate tubes at 37°C for 1 hour. Vortex gently every 15 minutes.
Sediment cysts again as described before.
Discard the supernatant and resuspend cells in 1.5 mL of fresh (made day of) Giardia media. Briefly vortex to mix cysts.
Incubate tubes at 37°C for 1 hour with periodic agitation.
Concentrate excyzoites at 135 × g for 5 min.
Aspirate supernatant and resuspend in Giardia media.
Count excyzoite numbers using a hemocytometer.
Excyzoites will appear motile.
BASIC PROTOCOL 3: IN VIVO INFECTIONS USING GIARDIA TROPHOZOITES
Much of what we know about the host-pathogen interaction between Giardia and the mammalian immune system is through the use of animal models. Giardia can infect mice in a laboratory setting; however, the intestinal microbiota can render mice resistant to colonization and must be accounted for during infection experiments. Mice purchased from Jackson Labs are typically susceptible to infection while those from Taconic Farms have frequently been resistant to infection. Treatment with antibiotic co*cktails can make resistant mice susceptible to colonization (). When analyzing transgenic lines of mice, including knockout strains, comparisons with littermate controls are essential to limit the impact of differing microbiotas on infection outcomes. The trophozoite form of Giardia is primarily used for the initiation of infection, however, cysts (purchased from Waterborne, Inc.) have also been used. It is unclear if in vitro generated cysts are infectious. Most infection studies have examined parasite burden at different times post-infection by direct counting of duodenal contents using a hemocytometer as described below. Recently, the use of in vivo imaging has been reported using strains of Giardia expressing luciferase enzymes (Barash et al., 2017). This can reduce the number of animals needed as euthanasia is not required to determine parasite burdens. In mice infected with trophozoites of strain GS, parasite burdens typically peak between 6–7 days post-infection with most parasites eliminated by day 14.
NOTE: Protocols using live animals must be reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and must adhere to government regulations regarding the use and care of animals. Additionally, all lab personnel must adhere to institutional training guidelines and rules when using the In Vivo Imaging System for live imaging.
Materials
Giardia trophozoites prepared in Basic Protocol 1 or Giardia cysts purchased from Waterbourne, Inc.
5 – 10-week-old C57BL/6J mice (Jackson Laboratory, Stock no. 000664)
Phosphate buffered saline (PBS) without calcium or magnesium
70% Ethanol
Isoflurane (Baxter Healthcare 1001936060)
Sterile D-luciferin solution (15 mg/mL) (PerkinElmer Part Number #122799)
In Vivo Imaging System (Perkin Elmer, IVIS)
Inverted microscope equipped with phase-contrast optics
Hemocytometer
20 G x 1”, 2.25 mm Animal Feeding Needle
1-mL syringe
Dissection Scissors
Dissection Forceps
35 mm Petri Dish
Murine Infections with Giardia
Work in BSC while wearing a protective laboratory coat and disposable examination gloves.
Clear workspace with disinfectant and move mouse cage into a BSC and set aside.
Resuspend trophozoites to a target concentration of 1 million trophozoites in 0.1 mL using sterile PBS.
Infectious dosage can vary by investigator but should be standardized between experiments.
Attach an oral gavage needle to a 1 mL syringe and load with an appropriate volume of trophozoites. Clear any trapped bubbles within syringes before administration.
Restrain mouse with proper scruffing technique and gently insert the feeding needle into the esophagus. If any resistance is felt, remove the needle, and try again as resistance may indicate improper placement in the trachea rather than the esophagus. Slowly administer the infectious dose and wait 2–3 seconds before removing the needle.
Release the newly infected mouse into a new cage to not mix up mice. Repeat step 5 until all designated mice are infected.
In vivo live imaging of Trophozoites (Barash et al., 2017)
After the desired number of days, shave the ventral abdomen of each mouse in preparation for live imaging.
For optimal detection of luciferin-expressing trophozoites, a luciferin kinetic curve should be performed in any new animal model before the collection of reportable data.
Sedate mice using isoflurane (1.5 to 3%) in an induction chamber.
Inject D-luciferin (15 mg/mL) intraperitoneally with each mouse receiving 0.15 mg luciferin/g of body weight and track time of injection.
The total injection volume is dependent on the weight of mice, but should not exceed 200 uL. Injections should be staggered so that every mouse reaches maximum luciferin signal during imaging.
Transfer sedated mice imager chamber and position mice on their dorsal surface (abdomen facing upward).
Maintain chamber anesthesia with 1.5 to 2% isoflurane and 100% oxygen during imaging.
Collect images with 2-minute exposures constantly over a time of 10 minutes.
The bioluminescent signal will become increasingly stronger over time as luciferin substrate disseminates. The maximum luciferase signal typically appears between 10–20 minutes after the injection of luciferin. It is important to compare signals obtained at the same time after luciferin injection.
The final image collection should use 2- or 5-minute exposures that are dependent on luciferase signal strength.
Detection of photons should be analyzed within the region of interest (abdomen or intestine) to quantify bioluminescence using image analysis computer programs.
Bioluminescence should be quantified as total flux (photons/second) and should be normalized to the background level of uninfected (non-luciferase expressing) mice.
Quantification of Trophozoites within the murine small intestine
After the desired number of days, euthanize infected mice using guidelines approved by your Institutional Animal Care and Use Committee. Spray mouse abdomen with 70% Ethanol to disinfect the dissection area.
Make a slight midline incision and retract the skin to expose peritoneum.
Cut peritoneum to expose the peritoneal cavity.
Make 2 individual cuts to excise a 3 cm segment of the upper small intestine (duodenum). One cut should be immediately distal to the ligament of Treitz (~2–3 cm below the stomach) and the second should be 3 cm from the initial cut. The ligament of Treitz is the point where the common bile duct enters the small intestine. Giardia require bile for growth and are typically more abundant just below this point.
Transfer the excised intestinal segment to a petri dish filled with 3 mL of sterile PBS.
The volume of PBS and the length of the intestinal segment being used for parasites should be equivalent to give a unit of trophozoites/cm of intestine.
Chill segments for 20–30 minutes on ice
Use curved scissors to finely mince the intestinal segment and gently swirl the dish to dissociate tissue that has aggregated.
Count trophozoites using a hemocytometer. It may take practice to count accurately since the trophozoites swim across the slide and attach to both the slide and the coverslip. Focusing up and down is required for accurate counting.
Pipette directly into the solution as trophozoites will detach from tissue after mincing. Avoid tissue clumps when pipetting as the tissue will clog pipette tip or give inaccurate cell counts.
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes, and protocol steps. If denoted otherwise, when media is not in use, store at 4°C. Bovine serum must be heat-inactivated by placing at 56°C for 30 minutes. Peptone digest, yeast extract, and bovine serum should be lot tested. All recipes can be down- or up-scaled according to need.
6.5% Bile Solution
6.5 g Bovine and Ovine Bile (Sigma Aldrich, cat. no. B8381)
100 mL Water
Filter sterilize with a 0.45 μM filter unit
Store at 4°C for up to 2 months.
2.28% Ferric Ammonium Citrate
2.28 g Ammonium iron (III) Citrate (Sigma Aldrich, cat. no. F5879)
100 mL Water
Filter sterilize with a 0.45 μM filter unit.
Store at 4°C for up to 2 months.
20X Lactic Acid
0.55 g L-lactic acid hemicalcium salt (Sigma-Aldrich, cat. no. L2000)
50 mL Water
Filter sterilize with a 0.45 μM filter unit
Must be made fresh and used immediately on the day of preparation.
40X Porcine Bile
1 g Porcine Bile (Sigma Aldrich, cat. no. B8631)
100 mL Water
Filter sterilize with a 0.45 μM filter unit
Must be made fresh and used immediately on the day of preparation.
Giardia Media (Complete Modified TYI-S-33 Medium)
Solution 1:
10 g Peptone from milk solids (Sigma-Aldrich, cat. no. P6838)
5 g D-glucose
5 g Bacto Yeast Extract (Gibco, cat. no. 212750)
1 g Sodium Chloride
1.05 g Sodium Bicarbonate
400 mL Water
4 mL Sterile 6.5% Bile Solution
1.5 mL Sterile 2.28% Ferric Ammonium Citrate
Alternatively, Sodium Bicarbonate can be substituted for 0.3 g of 3% KH2PO4 and 0.65 g K2HPO4•3H20.
As media components for Giardia media solution 1 solubilize and combine, media should turn from a somewhat opaque solution into a clear solution.
Solution 2:
1 g L-cysteine Hydrochloride (Sigma Aldrich, cat. no. C1276)
0.05 g Ascorbic Acid
30 mL Water
Adjust pH of solution 2 to 7.0–7.2 with 10N Sodium Hydroxide
Add solution 2 to solution 1 and mix for at least 20–30 minutes.
Add 50 mL Adult Bovine Serum and 5 mL Antibiotic-Antimycotic (Gibco, cat. no. 15240–062)
Sterile filter with 0.45 μm filter and aliquot in BSC (if needed).
Store up to 1–2 weeks at 4°C. Over time the cysteine in the media will oxidize to cystine and precipitate. Media should be discarded at this time and fresh media remade.
Encystation Medium #1
This recipe follows the Pre-encystation Medium with the following modification:
Adjust pH of solution 2 to 7.8 with 10M Sodium Hydroxide instead of 7.0–7.2
Solution 1:
10 g Peptone from milk solids (Sigma-Aldrich, cat. no. P6838)
5 g D-glucose
5 g Bacto Yeast Extract (Gibco, cat. no. 212750)
1 g Sodium Chloride
1.05 g Sodium Bicarbonate
400 mL Water
1.5 mL Sterile 2.28% Ferric Ammonium Citrate
Alternatively, Sodium Bicarbonate can be substituted for 0.3 g of 3% KH2PO4 and 0.65 g K2HPO4•3H20.
As media components for Giardia media solution 1 solubilize and combine, media should turn from a somewhat opaque solution into a clear solution.
Solution 2:
1 g L-cysteine Hydrochloride (Sigma Aldrich, cat. no. C1276)
0.05 g Ascorbic Acid
30 mL Water
Adjust pH of solution 2 to 7.8 with 10N Sodium Hydroxide
Add solution 2 to solution 1 and mix for at least 20–30 minutes
Add 50 mL Adult Bovine Serum and 5 mL Antibiotic-Antimycotic (Gibco, cat. no. 15240–062)
Sterile filter with 0.45 μm filter
Aseptically add 25 mL of sterile 40X Porcine bile
Aseptically 50 mL of sterile 20X lactic acid
This media must be made fresh and used immediately on the day of preparation.
Encystation Medium #2
This recipe follows the Pre-encystation Medium with the following modifications:
Add 6.5g Bovine and Ovine Bile to solution 1
Replace 1 g L-cysteine HCL in solution 2 with 1 g of L-cysteine (non HCL)
Adjust pH of solution 2 to 7.8 with 10N Sodium Hydroxide instead of pH 7.0–7.2
Solution 1:
10 g Peptone from milk solids (Sigma-Aldrich, cat. no. P6838)
5 g D-glucose
5 g Bacto Yeast Extract (Gibco, cat. no. 212750)
1 g Sodium Chloride
1.05 g Sodium Bicarbonate
6.5 g Bovine and Ovine Bile (Sigma Aldrich, cat. no. B8381)
400 mL Water
1.5 mL Sterile 2.28% Ferric Ammonium Citrate
Alternatively, Sodium Bicarbonate can be substituted for 0.3 g of 3% KH2PO4 and 0.65 g K2HPO4•3H20.
As media components for Giardia media solution 1 solubilize and combine, media should turn from a somewhat opaque solution into a clear solution.
Solution 2:
1 g L-cysteine (non HCL) (Sigma Aldrich, cat. no. C7352)
0.05 g Ascorbic Acid
30 mL Water
Adjust pH of solution 2 to 7.8 with 10M Sodium Hydroxide
Add solution 2 to solution 1 and mix for at least 20–30 minutes
Add 50 mL Adult Bovine Serum and 5 mL Antibiotic-Antimycotic (Gibco, cat. no. 15240–062)
Sterile filter with 0.45 μm filter
Aseptically add 50 mL of sterile 20X lactic acid
This media must be made fresh and used immediately on the day of preparation.
Excystation Stage 1 Solution
68 mg L-Cysteine (non HCL) (Sigma Aldrich, cat. no. C7352)
68 mg L-Glutathione Reduced (Sigma Aldrich, cat. no. G4251)
52 mg Sodium Bicarbonate
7 mL 1X Hank’s Balanced Salt Solution
18 mL Water
Adjust pH to 2.0 and sterilize with 0.45 μm filter
This media must be made fresh and used immediately on the day of preparation.
Excystation Stage 2 Solution
100 mg Trypsin Type II-S
10 mL 1X Tyrode’s Salt Solution, pH = 8
Mix and sterilize with 0.45 μm filter
This media must be made fresh and used immediately on day of preparation.
Pre-encystation Medium (Bile Starvation Medium)
This recipe follows the Giardia Media with the following modification:
Pre-encystation medium is not supplemented with 6.5% bile
Solution 1:
10 g Peptone from milk solids (Sigma-Aldrich, cat. no. P6838)
5 g D-glucose
5 g Bacto Yeast Extract (Gibco, cat. no. 212750)
1 g Sodium Chloride
1.05 g Sodium Bicarbonate
400 mL Water
1.5 mL Sterile 2.28% Ferric Ammonium Citrate
Alternatively, Sodium Bicarbonate can be substituted for 0.3 g of 3% KH2PO4 and 0.65 g K2HPO4•3H20.
As media components for Giardia media solution 1 solubilize and combine, media should turn from a somewhat opaque solution into a clear solution.
Solution 2:
1 g L-cysteine Hydrochloride (Sigma Aldrich, cat. no. C1276)
0.05 g Ascorbic Acid
30 mL Water
Adjust pH of solution 2 to 7.0–7.2 with 10N Sodium Hydroxide
Add 50 mL Adult Bovine Serum and 5 mL Antibiotic-Antimycotic (Gibco, cat. no. 15240–062)
Add solution 2 to solution 1 and mix for at least 20–30 minutes
Sterile filter with 0.45 μm filter
This media must be made fresh and used immediately on the day of preparation.
Trophozoite Freezing Medium A
4.5 mL Giardia Media
0.5 mL Sterile Fetal Bovine Serum (FBS)
Trophozoite Freezing Medium B
4.0 mL Giardia Media
0.5 mL Sterile Fetal Bovine Serum (FBS)
0.5 mL DMSO
COMMENTARY
Background Information
Giardia lamblia is a flagellated protozoan that is found ubiquitously throughout the world. Infection with Giardia in humans (giardiasis) is regarded as one of the most common causes of diarrheal disease, with an estimated 280 million cases reported worldwide each year (). Transmission of this parasite occurs via a fecal-oral route and is initiated through the ingestion of parasite contaminated water or food (Adam, 2001). The Giardia lifecycle has two distinct phases: a vegetative trophozoite that replicates in the lumen of the small intestine and an infective cyst that is resistant to harsh environmental conditions.
Monocultures of Giardia that were free from bacteria and yeast (axenic cultures) were first generated from the intestines of various animals (; Meyer, 1970). Axenic Giardia cultures were initially grown in complex HSP-1 medium (Meyer, 1970, 1976) and then transitioned to Diamond’s TP-S-1 medium (; Visvesvara, 1980). A modified recipe of TYI-S-33 medium then became the preferred medium for Giardia cultivation when it was modified with L-cysteine (, 1981b) and bile (Keister, 1983), as these reagents were able to better replicate conditions within the upper small intestine, Giardia’s environmental niche. Keister’s recipe has withstood the test of time and is still the basis for cultivating Giardia trophozoites in a laboratory setting.
Giardia is unique in that it one of the few protozoans that can be induced to differentiate into cysts using in vitro methods that replicate the host’s intestinal tract (). Each section of the intestine is important as the absorption of specific nutrients varies by intestinal location, thus, leading to differences in chemical composition between sections. Trophozoites that are swimming and migrating towards the colon with the host’s intestinal fluid can respond to this chemical change and are thought to encyst based on the changing availability of nutrients like bile and fatty acids. Accordingly, the majority of cysts in vivo are located distally within the gastrointestinal tract in the ileum, cecum, and colon (Gillin et al., 1987). As such, the two encystation protocols listed in this article induce encystation by a combination of pH adjustment, increased bile concentration leading to altered fatty acid availability, and the addition of lactic acid salts.
Giardia cysts are robust structures that are temperature resistant and can survive in water at 4°C for up to three months. Infected individuals can shed up to 10 billion cysts a day, and ingestion of as few as 10 cysts can initiate an infection (Rendtorff, 1954). Encystation specific vesicles (ESVs) are easily identifiable structures that form in trophozoites upon encystation induction after 8–24 hours in encystation media. ESVs appear as protuberances with Nomarski differential interference contrast (DIC) optics () or as dark spots under phase contrast (). However, viable cysts should also be resistant to water-treatment and also capable of excystation into trophozoites. Ultimately, the efficiency of each protocol will most likely vary between individual laboratories (discussed in Critical Parameters and Troubleshooting section).
Giardia cysts can be classified into two forms: type 1 and type 2. Type 1 cysts are considered to be the best quality for excystation. This type of cyst is oval and will appear bright under phase-contrast microscopy. The cyst wall and intracellular organelles are also clearly defined and easily identified. Additionally, cytosolic structures can be visualized inside the cyst and will be trypan-blue negative (white) (). Type 2 cysts are not as structured or defined as type 1 cysts. The cell body or cytoplasm does not fill up the entire cytosolic space and appears detached from the cyst wall and is trypan-blue positive (blue) ().
Most reports involving in vitro encystation protocols have been optimized for strain WB clone C6 (ATCC # 50803) and not strain GS (ATCC #50581). This may be problematic for in vivo studies as Giardia infection studies initiated by strain WB trophozoites are unable to properly colonize mice without prior antibiotic treatment to eliminate intestinal microbiota (). It is currently unknown if in vitro generated cysts of WB or GS strains are infective and truly replicate a meaningful biological host response. Animal infections initiated with Giardia cysts have used the H3 strain (Bartelt et al., 2017; Bartelt et al., 2013); however, these cysts have been purchased from Waterbourne, Inc as purified cysts that have been isolated from infected gerbils. As cysts are the natural infectious form of Giardia, it will be important to understand if host immune responses differ between the initiation of animal infections using cysts or trophozoites.
The Public Health Impact of Giardia
Developing countries that have poor water sanitation processes have a prevalence of giardiasis of 20% or greater (). In contrast, the United States, which has adequate water sanitation, has an estimated annual prevalence of <1% (Scallan et al., 2011). In areas endemic for giardiasis, Giardia was found to be the 4th most common pathogen found in stools of children younger than 1 year of age and the 2nd most common pathogen among children between 1 and 2 years of age (Platts-Mills et al., 2015).
Symptoms of giardiasis typically appear 6 – 15 days after infection (Farthing, 1997) and are characterized by diarrhea, intestinal cramps, nausea, intestinal malabsorption, and reduction in brush-border disaccharidases. Long-term manifestations have also been linked to Giardia, including irritable bowel and chronic fatigue syndrome. These can develop in giardiasis patients years after this parasite has been eliminated (Hanevik et al., 2017; Litleskare et al., 2018). Sub-clinical infections also appear to be very common in endemic areas which makes treatment difficult to initiate. Giardiasis is usually treatable with metronidazole or albendazole yet each has varying efficacies (Ansell et al., 2015; ; ) and vaccines are not currently available for human use ().
Unlike other intestinal infections, intestinal inflammation is typically not evident during giardiasis (Oberhuber et al., 1996). The Global Enteric Multicenter Study (GEMS) has reported that children infected with Giardia have a reduce incidence of severe pediatric diarrheal disease, suggesting the presence of this parasite may be protective through unknown mechanisms (Kotloff et al., 2013; ; ). On the other hand, The Etiology, Risk Factors, and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) project reported that Giardia detection was among the top five contributors to stunting (low height-for-age z-scores) in children worldwide (Rogawski et al., 2018). Additionally, this study found that cumulative parasite burdens were associated with childhood stunting, yet symptomatic diarrhea was not (Rogawski et al., 2018). The mechanisms by which repeated enteric infections lead to malnutrition and deficits in child development remain to be defined.
Giardia is detected using three diagnostic tests that require samples from suspected giardiasis patients. Traditional microscopy is based on the identification of microbes that appear to have the morphology of Giardia (). Light microscopy without the use of differential staining is unable to reveal this parasite to a species level () and staining protocols may involve iodine (), iron-hematoxylin (Garcia, 2009), Giemsa (Ament, 1972), or trichrome (; ). Immunological-based detection methods such as fluorescence assays or enzyme-linked immunosorbent assay (ELISA) that detect Giardia antigens (i.e. cyst wall proteins) can also be used to detected Giardia and provide a greater level of sensitivity and specificity when compared to microscopic techniques. Many commercialized detection tests for humans and animals are based on this method; however, protocols are not standardized and detection results may vary due to a lack of cross-reactivity between Giardia strains, quality of the sample, and subjectivity of sample results during analysis (; Koehler et al., 2014). These two detection methods are unable to discriminate Giardia based on a species and assemblage level which may become problematic during the detection of microbes within a large cohort as patients may be infected with mixed Giardia assemblages (). Polymerase chain reaction (PCR)-based techniques are the primary methods used to detect Giardia as it rapidly provides the greatest level of sensitivity and specificity. Additionally, these methods are also able to quantify the parasite burden of an infected individual; however, PCR techniques arguably require the greatest technical skill and are the most cost-prohibitive for detection ().
Critical Parameters and Troubleshooting
Giardia media is normally simple to make and should be fine to use for 7–10 days after creation as long as it is stored at 4˚C. Most difficulty in preparing Giardia media is typically due to manufacturer lot variability, clogging of vacuum filters due to precipitant formation, or the lack of recognizing expired media. Bovine serum and peptone vary by manufacturer and will not support trophozoite growth if a lot isn’t suitable for Giardia culture. Yeast extract also suffers from lot variation, although typically is more consistent. Reagents should first be batch tested to identify a usable lot for culturing and then purchased in bulk for long-term storage as peptone and frozen serum both have long shelf lives. Sterile filter vacuums can easily be clogged due to precipitant formation or from a lack of mixing. Polyethylene sulfonate filters are preferred in our lab. Also, our lab has modified Keister’s original recipe (Keister, 1983) by replacing the KH2PO4 and K2HPO4•3H2O with sodium bicarbonate as a pH buffer, due to phosphates propensity to precipitate from solution and consequently clog vacuum filters. Trophozoite cultures do not seem affected by this switch and remain viable. Additionally, it is critical to mix Giardia media for at least 20 minutes or until the solution turns clear as this will ensure that all components have been solubilized. The expiration of Giardia media is easily identified when white precipitate settles at the bottom of the bottle. This is due to the oxidation of cysteine and appears as hexagonal crystal under a microscope. At this point, the media is no good and needs to be thrown away. Keeping the media in a tightly-sealed bottle with little air prolongs the shelf-life. Giardia media is capable of being frozen at −20°C for extended periods, however, some batches of peptone will precipitate after freezing and thawing.
Giardia is considered a microaerophile and can tolerate small amounts of O2, however, large amounts of O2 (large open spaces) within the culture vessel will lead to a decrease in the viability of Giardia trophozoites. Thus, when growing Giardia in a culture vessel it is critical to leave little to no air within the vessel. Cell culture flasks with vented caps should never be used to culture Giardia and only plug-seal caps are suitable. Growing tubes at a slight angle also reduces the surface-air interface.
If Giardia is being cultivated in preparation for animal infections, then it is important to consider which strain will be used for infection. Giardia infection studies indicate that strain WB trophozoites are unable to properly colonize mice without prior antibiotic treatment (), while infections initiated by strain GS trophozoites or H3 cysts do not require prior antibiotic treatment. Prior treatment with antibiotics should be carefully considered, however, as depletion of the intestinal microbiota will alter the host’s immune response (). The interactions between Giardia and the intestinal microbiome are critical and have been summarized in Fink and Singer (2017).
Understanding Results
Live Giardia trophozoites will adhere to the bottom of the tube and also may be swimming or “spinning” in the supernatant (despite flowing with the movement of the liquid). Cells should appear to be teardrop in shape and not round. Under higher magnification, adherent cells can also be seen beating their flagella. Dead trophozoites will be floating with the movement of liquid and will not show any other movement. The rate of growth for Giardia trophozoites is dependent on the strain. Assemblage A (WB strain) of Giardia double every 6–8 hours, whereas assemblage B (GS or H3 strain), require 10–12 hours per doubling. All strains typically reach ~106 trophozoites/mL when confluent.
Cyst formation should begin with the addition of encystation medium; however, variability within an individual laboratory with regard to the efficiency of the encystation protocol used should be expected. Encysting trophozoites that produce encystation specific vesicles (ESVs) are good indicators that encystation machinery is active. Encysting cells will morph from teardrop shapes into ovals or circles that have a defined cyst wall.
Quantification of trophozoites from infected animals is highly variable and is dependent on infectious dose, mouse strain, and animal vendor. If parasites are viable and healthy, the peak burden of Giardia trophozoite will occur around day 6 or 7 in Giardia-infected C57BL/6J mice and the majority of parasites will be eliminated by day 14. In immunocompromised mice (e.g., SCID), Giardia-infections are unable to be controlled and parasite colonization will be extremely high throughout the infection. It is recommended that any mice used for Giardia infections are purchased from Jackson laboratories () and littermate controls should be used when appropriate.
Time Considerations
Adult bovine serum, peptone digest of casein, and yeast extract should all be lot tested first to identify a batch that is suitable for Giardia culture. The creation of all the media listed here should take approximately 30–60 minutes to complete. The 6.5% bile and 2.28% ferric ammonium citrate solutions for Giardia media and 20X lactic acid and 40X porcine bile solutions for encystation medium should be created first as all of these solutions are supplements. Thawing of Giardia trophozoites will take 5–10 minutes and the entire thawing procedure will take approximately 30–40 minutes. Generally, passaging and expanding trophozoite cultures can be accomplished within 45–60 minutes when using flask cultures, or within a shorter time of 15–20 minutes when using glass tubes. After chilling trophozoites, the freezing procedure should take approximately 20–40 minutes to complete. Cells being harvested to be used for either animal infections or cell culture assays are required to go through multiple washes to remove residual media; thus, harvesting of trophozoites will take between 1–2 hours depending on the number of washes conducted. All encystation procedures are simply just an exchange of media and should not take longer than 15 minutes to complete. The infection of mice is dependent on animal handling skills and the number of mice being infected; however, the infection of 10 mice should take 30–45 minutes. Quantification of parasites from infected mice is also dependent on the number of mice being analyzed with the imager and intestinal segments being assessed for parasites. After chilling of tissue, the completion of parasite counts for 1 mouse should take approximately 5–15 minutes. Time consideration for in vivo live imaging will vary due to experimental and imager setup, however, 2 or 3 mice may be able to be imaged concurrently. For a more accurate estimation of time, a luciferin kinetic curve should be performed in any new animal model.
Acknowledgements
The authors are supported by the National Institutes of Health grants AI-094492 and AI-109591 to SMS.
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